Deuterated compounds and uses thereof

ABSTRACT

The present invention provides for the treatment, prevention, and/or reduction of a risk of a disease, disorder, or condition in which aldehyde toxicity is implicated in the pathogenesis, including ocular disorders, skin disorders, conditions associated with injurious effects from blister agents, and autoimmune, inflammatory, neurological and cardiovascular diseases by the use of a primary amine to scavenge toxic aldehydes, such as MDA and HNE.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/720,645, filed Dec. 19, 2019; which is a continuation of U.S. patentapplication Ser. No. 15/754,065, filed Feb. 21, 2018, now U.S. Pat. No.10,550,085; which is a § 371 National Stage of PCT InternationalApplication No. PCT/US2016/048054, filed Aug. 22, 2016; which claims thebenefit of U.S. Provisional Patent Application No. 62/208,223, filedAug. 21, 2015. The entirety of each application is incorporated hereinby reference thereto.

BACKGROUND OF THE INVENTION

Metabolic and inflammatory processes in cells generate toxic aldehydes,such as malondialdehyde (MDA) and 4-hydroxyl-2-nonenal (HNE or 4HNE).These aldehydes are highly reactive to proteins, carbohydrates, lipidsand DNA, leading to chemically modified biological molecules, activationof inflammatory mediators such as NF-kappaB, and damage in diverseorgans. For example, retinaldehyde can react withphosphatidylethanolamine (PE) to form a highly toxic compound calledA2E, which is a component of lipofuscin believed to be involved in thedevelopment and progression of Age Related Macular Degeneration (AMD).Many bodily defense mechanisms function to remove or lower the levels oftoxic aldehydes. Novel small molecule therapeutics can be used toscavenge “escaped” retinaldehyde in the retina, thus reducing A2Eformation and lessening the risk of AMD (Jordan et al. (2006)).

Aldehydes are implicated in diverse pathological conditions such as dryeye, cataracts, keratoconus, Fuch's endothelial dystrophy in the cornea,uveitis, allergic conjunctivitis, ocular cicatricial pemphigoid,conditions associated with photorefractive keratectomy (PRK) healing orother corneal healing, conditions associated with tear lipid degradationor lacrimal gland dysfunction, inflammatory ocular conditions such asocular rosacea (with or without meibomian gland dysfunction), andnon-ocular disorders or conditions such as skin cancer, psoriasis,contact dermatitis, atopic dermatitis, acne vulgaris, Sjogren-LarssonSyndrome, ischemic-reperfusion injury, inflammation, diabetes,neurodegenerati on (e.g., Parkinson's disease), scleroderma, amyotrophiclateral sclerosis, autoimmune disorders (e.g., lupus), cardiovasculardisorders (e.g., atherosclerosis), and conditions associated with theinjurious effects of blister agents (Negre-Salvagre et al. (2008),Nakamura et al. (2007), Batista et al. (2012), Kenney et al. (2003), IntJ Dermatol 43: 494 (2004), Invest Ophthalmol Vis Sci 48: 1552 (2007),Graefe's Clin Exp Ophthalmol 233: 694 (1994), Molecular Vision 18: 194(2012)). Reducing or eliminating aldehydes should thus ameliorate thesymptoms and slow the progression of these pathological conditions.

MDA, HNE and other toxic aldehydes are generated by a myriad ofmetabolic mechanisms involving: fatty alcohols, sphingolipids,glycolipids, phytol, fatty acids, arachadonic acid metabolism (Rizzo(2007)), polyamine metabolism (Wood et al. (2006)), lipid peroxidation,oxidative metabolism (Buddi et al. (2002), Zhou et al. (2005)), andglucose metabolism (Pozzi et al. (2009)). Aldehydes can cross link withprimary amino groups and other chemical moieties on proteins,phospholipids, carbohydrates, and DNA, leading in many cases to toxicconsequences, such as mutagenesis and carcinogenesis (Marnett (2002)).MDA is associated with diseased corneas, keratoconus, bullous and otherkeratopathy, and Fuch's endothelial dystrophy corneas (Buddi et al.(2002)). Also, skin disorders, e.g., Sjogren-Larsson Syndrome, arelikely connected with the accumulation of fatty aldehydes such asoctadecanal and hexadecanal (Rizzo et al. (2010)). Further, increasedlipid peroxidation and resultant aldehyde generation are associated withthe toxic effects of blister agents (Sciuto et al. (2004) and Pal et al.(2009)).

There has been no suggestion in the art for treating the variousconditions associated with toxic aldehydes by the administration ofsmall molecule therapeutics acting as a scavenger for aldehydes, such asMDA and/or HNE. Thus, there is a need for treating, preventing, and/orreducing a risk of a disease or disorder in which aldehyde toxicity isimplicated in the pathogenesis. The present invention addresses such aneed.

Accordingly, there remains a need for treating, preventing, and/orreducing a risk of a disease, disorder, or condition in which aldehydetoxicity is implicated in the pathogenesis.

SUMMARY OF THE INVENTION

It has now been found that compounds of the present invention, andcompositions thereof, are useful for treating, preventing, and/orreducing a risk of a disease, disorder, or condition in which aldehydetoxicity is implicated in the pathogenesis. Such compounds have generalformula I:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is as defined herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with toxic aldehydes. Such diseases,disorders, or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofcertain aldehydes in biology and pathological phenomena.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows overlays of EICs (extracted ion chromatograms) of themetabolite profiles of NS2 at 0 min and 120 min in human hepatocytescompared with a reference (blank hepatocytes). As the overlays show,after 120 min NS2 is metabolized to M1, M7, M8, and a low amount of M9,with some unchanged NS2 remaining.

FIG. 2 shows overlays of EICs of the metabolite profiles of NS2 at 0 minand 120 min in monkey hepatocytes compared with a reference (blankhepatocytes). As the overlays show, after 120 min NS2 is metabolized toM1, M3, M4, M7, M8, and a low amount of M9, with some unchanged NS2remaining.

FIG. 3 shows overlays of EICs of the metabolite profiles of NS2 at 0 minand 120 min in dog hepatocytes compared with a reference (blankhepatocytes). As the overlays show, after 120 min NS2 is metabolized toM1, M2, M5, and M6, with some unchanged NS2 remaining.

FIG. 4 shows overlays of EICs of the metabolite profiles of NS2 at 0 minand 120 min in rat hepatocytes compared with a reference (blankhepatocytes). As the overlays show, after 120 min NS2 is metabolized toM1, M7, and M8, with some unchanged NS2 remaining.

FIG. 5 shows overlays of EICs of the metabolite profiles of DeuteratedNS2 (NS2-D6; compound I-1) at 0 min and 120 min in human hepatocytescompared with a reference (blank hepatocytes). As the overlays show,after 120 min NS2-D6 is metabolized to a small amount of M1, with mostlyunchanged NS2-D6 remaining.

FIG. 6 shows mass spectral analysis of NS2 (m/z=237).

FIG. 7 shows mass spectral analysis of metabolite M1 (m/z=253, RT=2.1min).

FIG. 8 shows mass spectral analysis of metabolite M2 (m/z=253, RT=2.9min).

FIG. 9 shows mass spectral analysis of metabolite M3 (m/z=429, RT=3.0min).

FIG. 10 shows mass spectral analysis of metabolite M4 (m/z=429, RT=3.2min).

FIG. 11 shows mass spectral analysis of metabolite M5 (m/z=542, RT=3.5min).

FIG. 12 shows mass spectral analysis of metabolite M6 (m/z=542, RT=3.7min). Note: m/z of 413 represents a neutral loss (NL) of 129, indicativeof GSH fragmentation pattern.

FIG. 13 shows mass spectral analysis of metabolite M7 (m/z=429, RT=3.7min).

FIG. 14 shows mass spectral analysis of metabolite M8 (m/z=413, RT=3.9min).

FIG. 15 shows mass spectral analysis of metabolite M9 (m/z=429, RT=3.9min).

FIG. 16 shows mass spectral analysis of NS2-D6 (compound I-1; m/z=243).

FIG. 17 shows mass spectral analysis of metabolite 1 of NS2-D6(m/z=259).

FIG. 18 shows the effect of NS2 (CoreRx) on neuronal viability afterco-treatment with 10 μM hydrogen peroxide for 5 hours.

FIG. 19 shows a graph fitting the CFDA (in relative fluorescence units)data at varying NS2 (CoreRx; in Captisol®) concentrations to a curvefrom which is derived the EC₅₀ value.

FIG. 20 shows the effect of NS2 (CoreRx) on cell death in hippocampalcultures after co-treatment with 10 μM hydrogen peroxide for 5 hours.*indicates data points that are significantly different from HPtreatment alone.

FIG. 21 shows a graph fitting the propidium iodide data (in relativefluorescence units) at varying NS2 (CoreRx; in Captisol®) concentrationsto a curve from which the EC₅₀ value is derived.

FIG. 22 shows the effect of NS2 (CoreRx; in DMSO) on neuronal viabilityafter co-treatment with 10 mM hydrogen peroxide for 5 hours. *indicatesdata points that are significantly different from HP treatment alone.

FIG. 23 shows a graph fitting the CFDA (in relative fluorescence units)data at varying NS2 (CoreRx; in DMSO) concentrations to a curve fromwhich is derived the EC₅₀ value.

FIG. 24 shows the effect of NS2 (CoreRx; in DMSO) on cell death inhippocampal cultures after co-treatment with 10 μM hydrogen peroxide.*indicates data points that are significantly different from HPtreatment alone.

FIG. 25 shows a graph fitting the propidium iodide date (in relativefluorescence units) at varying NS2 (CoreRx; in DMSO) concentrations to acurve from which the EC₅₀ value is derived.

FIG. 26 shows dose response data for non-milled NS2 (J-Star) inCaptisol® demonstrating the effect on neuronal viability afterco-treatment with 10 μM hydrogen peroxide. *indicates data points thatare significantly different from HP treatment alone.

FIG. 27 shows a graph fitting the CFDA (in relative fluorescence units)data at varying NS2 (J-Star; in DMSO) concentrations to a curve fromwhich is derived the EC₅₀ value.

FIG. 28 shows dose response data for non-milled NS2 (J-Star) inCaptisol® demonstrating the effect on cell death in hippocampal culturesafter co-treatment with 10 μM hydrogen peroxide. *indicates data pointsthat are significantly different from HP treatment alone.

FIG. 29 shows a graph fitting the propidium iodide data (in relativefluorescence units) at varying NS2 (non-milled (J-Star) in Captisol®)concentrations to a curve from which the EC₅₀ value is derived.

FIG. 30 shows dose response data for non-milled NS2 (J-Star) in DMSOdemonstrating the effect on neuronal viability in hippocampal culturesafter co-treatment with 10 μM hydrogen peroxide. *indicates data pointsthat are significantly different from HP treatment alone.

FIG. 31 shows a graph fitting the CFDA (in relative fluorescence units)data at varying non-milled NS2 ((J-Star) in DMSO) concentrations to acurve from which is derived the EC₅₀ value.

FIG. 32 shows dose response data for non-milled NS2 (J-Star) in DMSOshowing effect on cell death in hippocampal cells after co-treatmentwith 10 μM hydrogen peroxide. *indicates data points that aresignificantly different from HP treatment alone.

FIG. 33 shows a graph fitting the propidium iodide data (in relativefluorescence units) at varying non-milled NS2 ((J-Star) in DMSO)concentrations to a curve from which the EC₅₀ value is derived.

FIG. 34 shows dose response data for the formulation vehicles onneuronal cell viability after co-treatment with 10 μM hydrogen peroxide.

FIG. 35 shows dose response data for the formulation vehicles on celldeath after co-treatment with 10 μM hydrogen peroxide.

FIG. 36 shows the effect of ALD-6 (compound I-1) on neuronal viabilityin hippocampal cultures treated with 10 μM hydrogen peroxide. *indicates data points that are significantly different from HP treatmentalone.

FIG. 37 shows the calculated logistic curve and EC₅₀ value for neuronalviability assay using hippocampal cultures treated with ALD-6.

FIG. 38 shows dose response effect of ALD-6 on cell death afterco-treatment with 10 μM hydrogen peroxide. *indicates data points thatare significantly different from HP treatment alone.

FIG. 39 shows the calculated logistic curve and EC₅₀ value for celldeath assay using hippocampal cultures treated with ALD-6.

FIG. 40 shows dose response effect of ALD-5 on neuronal viability afterco-treatment with 10 μM hydrogen peroxide.

FIG. 41 shows dose response effect of ALD-5 on cell death afterco-treatment with 10 μM hydrogen peroxide.

FIG. 42 shows dose response effect of ALD-2 on neuronal viability afterco-treatment with 10 μM hydrogen peroxide.

FIG. 43 shows dose response effect of ALD-2 on cell death afterco-treatment with 10 μM hydrogen peroxide.

FIGS. 44-46 show a histogram of specific binding results for NS2-D6expressed as a percentage of the specific binding of each controlcompound.

FIG. 47 shows a histogram of in vitro pharmacology results in enzyme anduptake assays for NS2-D6.

DETAILED DESCRIPTION OF THE INVENTION 1. General Description of CertainAspects of the Invention

In certain embodiments, the present invention provides compounds,compositions, and methods for treatment, prevention, and/or reduction ofa risk of diseases, disorders, or conditions in which aldehyde toxicityis implicated in the pathogenesis. In some embodiments, such compoundsinclude those of the formulae described herein, or a pharmaceuticallyacceptable salt thereof, wherein each variable is as defined herein anddescribed in embodiments. In one aspect, the present invention providesa compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;    -   R² is selected from hydrogen or deuterium;    -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or        —CD₃; and    -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen        or deuterium; provided that at least one of R¹, R², R³, R⁴, R⁵,        R⁶, R⁷, or R⁸ is or contains deuterium.

2. Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representative alkalior alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.

The “retina” is a region of the central nervous system withapproximately 150 million neurons. It is located at the back of the eyewhere it rests upon a specialized epithelial tissue called retinalpigment epithelium (RPE). The retina initiates the first stage of visualprocessing by transducing visual stimuli in specialized neurons called“photoreceptors”. Their synaptic outputs are processed by elaborateneural networks in the retina and are then transmitted to the brain. Theretina has evolved two specialized classes of photoreceptors to operateunder a wide range of light conditions. “Rod” photoreceptors transducevisual images under low light conditions and mediate achromatic vision.“Cone” photoreceptors transduce visual images in dim to bright lightconditions and mediate both color vision and high acuity vision.

Every photoreceptor is compartmentalized into two regions called the“outer” and “inner” segment. The inner segment is the neuronal cell bodycontaining the cell nucleus. The inner segment survives for a lifetimein the absence of retinal disease. The outer segment is the region wherethe light sensitive visual pigment molecules are concentrated in a densearray of stacked membrane structures. Part of the outer segment isroutinely shed and regrown in a diurnal process called outer segmentrenewal. Shed outer segments are ingested and metabolized by RPE cells.

The “macula” is the central region of the retina which contains thefovea where visual images are processed by long slender cones in highspatial detail (“visual acuity”). “Macular degeneration” is a form ofretinal neurodegeneration which attacks the macula and destroys highacuity vision in the center of the visual field. Age-Related MacularDegeneration (AMD) begins in a “dry form” characterized by residuallysosomal granules called lipofuscin in RPE cells, and by extracellulardeposits called “drusen”. Drusen contain cellular waste productsexcreted by RPE cells. “Lipofuscin” and drusen can be detectedclinically by ophthalmologists and quantified using fluorescencetechniques. They can be the first clinical signs of maculardegeneration.

Lipfuscin contains aggregations of A2E. Lipofuscin accumulates in RPEcells and poisons them by multiple known mechanisms. As RPE cells becomepoisoned, their biochemical activities decline and photoreceptors beginto degenerate. Extracellular drusen may further compromise RPE cells byinterfering with their supply of vascular nutrients. Drusen also triggerinflammatory processes, which lead to choroidal neovascular invasions ofthe macula in one patient in ten who progresses to wet form AMD. Boththe dry form and wet form progress to blindness.

“ERG” is an acronym for electroretinogram, which is the measurement ofthe electric field potential emitted by retinal neurons during theirresponse to an experimentally defined light stimulus. ERG is anon-invasive measurement which can be performed on either livingsubjects (human or animal) or a hemisected eye in solution that has beenremoved surgically from a living animal.

As used herein, the term “RAL” means retinaldehyde. The term “RAL-trap”means a therapeutic compound that binds free RAL and thereby preventsthe RAL from Schiff base condensation with membranephosphatidylethanolamine (PE). “Free RAL” is defined as RAL that is notbound to a visual cycle protein. The terms “trans-RAL” and“all-trans-RAL” are used interchangeably and mean alltrans-retinaldehyde.

A2E is a reaction by-product of a complex biochemical pathway called the“visual cycle” which operates collaboratively in both RPE cells andphotoreceptor outer segments. The visual cycle recycles a photoreactivealdehyde chromophore called “retinaldehyde” which is derived fromvitamin A and is essential for vision. In simplified terms, the visualcycle has four principal steps: 1) it converts vitamin A in the RPE intoan aldehyde chromophore with one photoreactive strained double bond(11-cis-RAL); 2) it transports 11-cis-RAL to the retina where it bindsto a specialized photoreceptor protein called opsin; 3) lightphotoisomerizes bound 11-cis-RAL to trans-RAL, which initiates therelease of bound RAL from the opsin binding site; and 4) it convertstrans-RAL (an aldehyde) to vitamin A (an alcohol) and transports vitaminA back to the RPE where the cycle begins again.

The aldehyde group of RAL helps bind the molecule to opsin by forming areversible chemical bond to an amino acid sidechain in the opsin bindingsite. While the aldehyde group on RAL is essential for anchoring themolecule to the opsin binding site, it is otherwise hazardous because ofits propensity to form Schiff bases with other biological amines. Thefirst three reactions take place in photoreceptor outer segments andproduce an intermediary product called A2PE. Once formed, A2PEpartitions into the lipid phase and accumulates in photoreceptor outersegment membranes. When RPE cells ingest discarded outer segments, theiraccumulated A2PE is routed to their lysosomes.

As described above, macular degeneration and other forms of retinaldisease whose etiology involves the accumulation of A2E and/orlipofuscin may be treated or prevented by lowering the amount of A2Eformed. Compounds useful for doing so include RAL-traps. RAL-traps lowerthe amount of A2E formed, for example by forming a covalent bond withRAL that has escaped sequestering. RAL that has reacted with a RAL-trapcompound is thereby unavailable to react with phosphatidyl ethanolamine.

The present invention is also directed to the use of a compounddescribed herein in the manufacture of a medicament for the treatment,prevention, and/or reduction of a risk of a disease, disorder, orcondition in which aldehyde toxicity is implicated in the pathogenesis.More specifically this aspect of the invention is directed to the use ofa compound described herein in the manufacture of a medicament for thetreatment, prevention, and/or reduction of a risk of (1) an oculardisease, disorder, or condition, including, but not limited to, acorneal disease (e.g., dry eye syndrome, cataracts, keratoconus, bullousand other keratopathy, and Fuch's endothelial dystrophy), other oculardisorders or conditions (e.g., allergic conjunctivitis, ocularcicatricial pemphigoid, conditions associated with PRK healing and othercorneal healing, and conditions associated with tear lipid degradationor lacrimal gland dysfunction), and other ocular conditions associatedwith high aldehyde levels as a result of inflammation (e.g., uveitis,scleritis, ocular Stevens Johnson Syndrome, and ocular rosacea (with orwithout meibomian gland dysfunction)), (2) a skin disorder or conditionor a cosmetic indication. For example, the disease, disorder, orcondition includes, but is not limited to, psoriasis, topical (discoid)lupus, contact dermatitis, atopic dermatitis, allergic dermatitis,radiation dermatitis, acne vulgaris, Sjogren-Larsson Syndrome and otherichthyosis, solar elastosis/wrinkles, skin tone firmness, puffiness,eczema, smoke or irritant induced skin changes, dermal incision, and askin condition associated burn and wound, (3) a condition associatedwith the toxic effects of blister agents or burns from alkali agents, or(4) an autoimmune, immune-mediated, inflammatory, cardiovascular, orneurological disease (e.g., lupus, scleroderma, asthma, chronicobstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatorybowel disease, sepsis, atherosclerosis, ischemic-reperfusion injury,Parkinson's disease, Alzheimer's disease, multiple sclerosis,amyotrophic lateral sclerosis, diabetes, metabolic syndrome, andfibrotic diseases).

The present invention is also directed to the use of a compounddescribed herein in treating, preventing, and/or reducing a risk of adisease, disorder, or condition in which aldehyde toxicity is implicatedin the pathogenesis. More specifically this aspect of the invention isdirected to the use of a compound described herein in treating,preventing, and/or reducing a risk of (1) an ocular disease, disorder,or condition, including, but not limited to, a corneal disease (e.g.,dry eye syndrome, cataracts, keratoconus, bullous and other keratopathy,and Fuch's endothelial dystrophy), other ocular disorders or conditions(e.g., allergic conjunctivitis, ocular cicatricial pemphigoid,conditions associated with PRK healing and other corneal healing, andconditions associated with tear lipid degradation or lacrimal glanddysfunction), and other ocular conditions associated with high aldehydelevels as a result of inflammation (e.g., uveitis, scleritis, ocularStevens Johnson Syndrome, and ocular rosacea (with or without meibomiangland dysfunction)), (2) a skin disorder or condition or a cosmeticindication. For example, the disease, disorder, or condition includes,but is not limited to, psoriasis, topical (discoid) lupus, contactdermatitis, atopic dermatitis, allergic dermatitis, radiationdermatitis, acne vulgaris, Sjogren-Larsson Syndrome and otherichthyosis, solar elastosis/wrinkles, skin tone firmness, puffiness,eczema, smoke or irritant induced skin changes, dermal incision, and askin condition associated burn and wound, (3) a condition associatedwith the toxic effects of blister agents or burns from alkali agents, or(4) an autoimmune, immune-mediated, inflammatory, cardiovascular, orneurological disease (e.g., lupus, scleroderma, asthma, chronicobstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatorybowel disease, sepsis, atherosclerosis, ischemic-reperfusion injury,Parkinson's disease, Alzheimer's disease, multiple sclerosis,amyotrophic lateral sclerosis, diabetes, metabolic syndrome, andfibrotic diseases).

The compounds described herein can also be administered topically, suchas directly to the eye, e.g., as an eye-drop or ophthalmic ointment. Eyedrops typically comprise an effective amount of at least one compounddescribed herein and a carrier capable of being safely applied to aneye. For example, the eye drops are in the form of an isotonic solution,and the pH of the solution is adjusted so that there is no irritation ofthe eye. In many instances, the epithelial barrier interferes withpenetration of molecules into the eye. Thus, most currently usedophthalmic drugs are supplemented with some form of penetrationenhancer. These penetration enhancers work by loosening the tightjunctions of the most superior epithelial cells (Burstein, TransOphthalmol Soc UK 104: 402 (1985); Ashton et al., J Pharmacol Exp Ther259: 719 (1991); Green et al., Am J Ophthalmol 72: 897 (1971)). The mostcommonly used penetration enhancer is benzalkonium chloride (Tang etal., J Pharm Sci 83: 85 (1994); Burstein et al, Invest Ophthalmol VisSci 19: 308 (1980)), which also works as preservative against microbialcontamination.

Topical administration may be in the form of a cream, suspension,emulsion, ointment, drops, oil, lotion, patch, tape, inhalant, spray, orcontrolled release topical formulations including gels, films, patches,and adhesives. Intra-ocular administration may take the form ofsubconjunctival, subtenon's capsule, retrobulbar or intravitrealinjections, depots or implants. Compounds administered by these routesmay be in solution or suspension form. Administration of compounds bydepot injection may contain pharmaceutically acceptable carriers orexcipients; these may be natural or synthetic and may be biodegradableor non-biodegradable and facilitate drug release in a controlled manner.Implants used for controlled release of compound may be composed ofnatural or synthetic, biodegradable or non-biodegradable materials. Thecarrier is acceptable in that it is compatible with the other componentsof the composition and is not injurious to the patient. Some examples ofcarriers include (1) sugars such as lactose glucose and sucrose, (2)starches such as corn starch and potato starch, (3) cellulose and (4)cyclodextrins. A useful topical formulation is described in PCTpublication WO 2011/072141, the contents of which are hereinincorporated by reference.

Formulations for topical administration to the skin can include, forexample, ointments, creams, gels and pastes comprising the primary aminecompound in a pharmaceutical acceptable carrier. The formulation of theprimary amine compound for topical use includes the preparation ofoleaginous or water-soluble ointment bases, as is well known to those inthe art. For example, these formulations may include vegetable oils,animal fats, and, for example, semisolid hydrocarbons obtained frompetroleum. Particular components used may include white ointment, yellowointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum,white petrolatum, spermaceti, starch glycerite, white wax, yellow wax,lanolin, anhydrous lanolin and glyceryl monostearate. Variouswater-soluble ointment bases may also be used, including glycol ethersand derivatives, polyethylene glycols, polyoxyl 40 stearate andpolysorbates.

The formulations for topical administration may contain the compoundused in the present application at a concentration in the range of0.001-10%, 0.05-10%, 0.1-10%, 0.2-10%, 0.5-10%, 1-10%, 2-10%, 3-10%,4-10%, 5-10%, or 7-10% (weight/volume), or in the range of 0.001-2.0%,0.001-1.5%, or 0.001-1.0%, (weight/volume), or in the range of0.05-2.0%, 0.05-1.5%, or 0.05-1.0%, (weight/volume), or in the range of0.1-5.0%, 0.1-2.0%, 0.1-1.5%, or 0.1-1.0% (weight/volume), or in therange of 0.5-5.0%, 0.5-2.0%, 0.5-1.5%, or 0.5-1.0% (weight/volume), orin the range of 1-5.0%, 1-2.0%, or 1-1.5% (weight/volume). Theformulations for topical administration may also contain the compoundused in the present application at a concentration in the range of0.001-2.5%, 0.01-2.5%, 0.05-2.0%, 0.1-2.0%, 0.2-2.0%, 0.5-2.0%, or1-2.0% (weight/weight), or in the range of 0.001-2.0%, 0.001-1.5%,0.001-1.0%, or 0.001-5% (weight/weight).

In an eye drop formulation the composition may contain the activecompound at a concentration of 0.01-20%, 0.02-15%, 0.04-10%, 0.06-5%,0.08-1%, or 0.09-0.5% (weight/volume) with or without pH and/or osmoticadjustment to the solution. More particularly, the eye drop formulationmay contain a compound described herein at a concentration of 0.09-0.5%(weight/volume), such as 0.1%.

In one exemplification, the pharmaceutical compositions encompass acomposition made by admixing a therapeutically effective amount of acompound described herein with an oligomeric or a polymeric carrier suchas a cyclodextrin, or chemically modified cyclodextrin, includingtrimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin, andβ-cyclodextrin sulfobutylether sodium salt (or potassium salt).Exemplifying an oligomeric or a polymeric carrier is β-cyclodextrinsulfobutylether sodium salt. The amount of β-cyclodextrinsulfobutylether sodium salt in the composition may range from about0.01% to 30% weight/volume. In one illustration, the concentration ofβ-cyclodextrin sulfobutylether sodium salt is 5-25% weight/volume.Further illustrating the concentration of β-cyclodextrin sulfobutylethersodium salt is 6-20% weight/volume. In one exemplification theconcentration of β-cyclodextrin sulfobutylether is 6-12% weight/volume.Further exemplifying the concentration of β-cyclodextrin sulfobutyletheris 9-10% weight/volume, including 9.5% weight/volume. The amount of thecompound described herein in the composition may range 0.01-20%,0.02-15%, 0.04-10%, 0.06-5%, 0.08-1%, or 0.09-0.5% (weight/volume). Moreparticularly, the composition may contain a compound described herein ata concentration of 0.09-0.5% (weight/volume), such as 0.1%.

The compounds described herein may be administered orally and as suchthe pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations.

For oral administration in the form of a tablet or capsule (e.g., agelatin capsule), the active drug component can be combined with anoral, non-toxic pharmaceutically acceptable inert carrier such asethanol, glycerol, water and the like. Moreover, when desired ornecessary, suitable binders, lubricants, disintegrating agents andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, magnesium aluminum silicate, starch paste,gelatin, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, natural sugars such as glucose or beta-lactose,corn sweeteners, natural and synthetic gums such as acacia, tragacanthor sodium alginate, polyethylene glycol, waxes and the like. Lubricantsused in these dosage forms include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride,silica, talcum, stearic acid, its magnesium or calcium salt and/orpolyethyleneglycol and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gumstarches, agar, alginic acid or its sodium salt, or effervescentmixtures, croscarmellose or its sodium salt, and the like. Diluents,include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, celluloseand/or glycine.

Tablets contain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period.

A therapeutically effective dose, of a compound described herein in anoral formulation, may vary from 0.01 mg/kg to 50 mg/kg patient bodyweight per day, more particularly 0.01 to 10 mg/kg, which can beadministered in single or multiple doses per day. For oraladministration the drug can be delivered in the form of tablets orcapsules containing 1 mg to 500 mg of the active ingredientspecifically, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 250 mg, and 500mg, or in the forms of tables or capsules containing at least 1%, 2%,5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% (w/w) of the active ingredient.For example, the capsules may contain 50 mg of the active ingredient, or5-10% (w/w) of the active ingredient. For example, the tablets maycontain 100 mg of the active ingredient, or 20-50% (w/w) of the activeingredient. For example, the tablet may contain, in addition to theactive ingredient, a disintegrant (e.g., croscarmellose or its sodiumsalt and methyl cellulose), a diluent (e.g., microcrystallinecellulose), and a lubricant (e.g., sodium stearate and magnesiumstearate). The drug can be administered on a daily basis either once,twice or more per day.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art.

Parenteral formulations comprising a compound described herein can beprepared in aqueous isotonic solutions or suspensions, and suppositoriesare advantageously prepared from fatty emulsions or suspensions. Theformulations may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmethods, and may contain about 0.1 to 75%, preferably about 1 to 50%, ofa compound described herein.

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and include,without limitation, intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

3. Description of Exemplary Compounds

Deuterium (D or ²H) is a stable, non-radioactive isotope of hydrogen andhas an atomic weight of 2.0144. Hydrogen naturally occurs as a mixtureof the isotopes ¹H (hydrogen or protium), D (²H or deuterium), and T (³Hor tritium). One skilled in the art appreciates that the designation“hydrogen” in hydrogen-containing chemical compounds actually representsa mixture of hydrogen and about 0.015% deuterium.

Complete deuteration, or 100% deuteration, at any one site can bedifficult to achieve in the laboratory. When a deuterium atom isindicated at a given site on any compound described herein, it isunderstood that a small percentage of hydrogen may still be present.Such compounds are said to be enriched with deuterium.Deuterium-enriched compounds are prepared via synthesis utilizingappropriately enriched starting materials. As used herein, the terms“deuterium-enriched” or “deuterium enrichment” refer to a compound, or aparticular site of said compound, which comprises deuterium in an amountthat is greater than its natural isotopic abundance (0.015%).Accordingly, in some embodiments, the present invention providescompounds comprising deuterium at a given site, wherein the percentageor level of deuterium incorporation is greater than its natural isotopicabundance.

According to one aspect, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:R¹ is selected from —NH₂, —NHD, or —ND₂;R² is selected from hydrogen or deuterium;R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;andR⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, orR⁸ is or contains deuterium.

According to another aspect, the present invention provides a compoundof formula I-A:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, orR⁸ is or contains deuterium.

According to another aspect, the present invention provides a compoundof formulae II-A or II-B:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, orR⁸ in formula II-A is or contains deuterium.

According to another aspect, the present invention provides a compoundof formulae III-A, III-B, or III-C:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, orR⁸ is or contains deuterium.

According to another aspect, the present invention provides a compoundof formula IV:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, orR⁸ is or contains deuterium.

According to another aspect, the present invention provides a compoundof formula V:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, orR⁸ is or contains deuterium.

According to another aspect, the present invention provides a compoundof formulae VI-A or VI-B:

or a pharmaceutically acceptable salt thereof, wherein:

each A is independently hydrogen or deuterium;

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium; and

R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of A, R¹, R², R⁵, R⁶, R⁷, or R⁸ isor contains deuterium.

According to another aspect, the present invention provides a compoundof formulae VII-A or VII-B:

or a pharmaceutically acceptable salt thereof, wherein:

each A is independently hydrogen or deuterium;

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁸ is selected from hydrogen or deuterium;

provided that at least one of R¹, R², R³, R⁴, A, or R⁸ is or containsdeuterium.

According to another aspect, the present invention provides a compoundof formulae VIII-A or VIII-B:

or a pharmaceutically acceptable salt thereof, wherein:

each A is independently hydrogen or deuterium;

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵ and R⁸ are each independently selected from hydrogen or deuterium;

provided that at least one of A, R¹, R², R³, R⁴, R⁵, or R⁸ is orcontains deuterium.

According to another aspect, the present invention provides a compoundof formula IX-A or IX-B:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen ordeuterium; provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, orR⁸ is or contains deuterium.

According to another aspect, the present invention provides a compoundof formula X:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from —NH₂, —NHD, or —ND₂;

R² is selected from hydrogen or deuterium;

R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃;and

R⁵ and R⁶ are each independently selected from hydrogen or deuterium;

provided that at least one of R¹, R², R³, R⁴, R⁵, or R⁶ is or containsdeuterium.

The following embodiments are applicable to each of the precedingformulae.

As defined above and described herein, R¹ is selected from —NH₂, —NHD,or —ND₂.

In some embodiments, R¹ is —NH₂. In some embodiments, R¹ is —NH₂ and atleast one of R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R¹ is —NHD. In some embodiments, R¹ is —NHD and atleast one of R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R¹ is —ND₂. In some embodiments, R¹ is —ND₂ and atleast one of R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, A is selected from hydrogen ordeuterium.

In some embodiments, A is hydrogen. In some embodiments, A is hydrogenand at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or containsdeuterium. In some embodiments, A is deuterium. In some embodiments, Ais deuterium and at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R¹ is orcontains deuterium.

As defined above and described herein, R² is selected from hydrogen ordeuterium.

In some embodiments, R² is hydrogen. In some embodiments, R² is hydrogenand at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or containsdeuterium. In some embodiments, R² is deuterium. In some embodiments, R²is deuterium and at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is orcontains deuterium.

As defined above and described herein, R³ is selected from —CH₃, —CH₂D,—CHD₂, or —CD₃.

In some embodiments, R³ is —CH₃. In some embodiments, R³ is —CH₃ and atleast one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R³ is —CH₂D. In some embodiments, R³ is —CH₂D andat least one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R³ is —CHD₂. In some embodiments, R³ is —CHD₂ andat least one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R³ is —CD₃. In some embodiments, R³ is —CD₃ and atleast one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R⁴ is selected from —CH₃, —CH₂D,—CHD₂, or —CD₃.

In some embodiments, R⁴ is —CH₃. In some embodiments, R⁴ is —CH₃ and atleast one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R⁴ is —CH₂D. In some embodiments, R⁴ is —CH₂D andat least one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R⁴ is —CHD₂. In some embodiments, R⁴ is —CHD₂ andat least one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R⁴ is —CD₃. In some embodiments, R⁴ is —CD₃ and atleast one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R⁵ is selected from hydrogen ordeuterium.

In some embodiments, R⁵ is hydrogen. In some embodiments, R⁵ is hydrogenand at least one of R¹, R², R³, R⁴, R⁶, R⁷, or R⁸ is or containsdeuterium. In some embodiments, R⁵ is deuterium. In some embodiments, R⁵is deuterium and at least one of R¹, R², R³, R⁴, R⁶, R⁷, or R⁸ is orcontains deuterium.

As defined above and described herein, R⁶ is selected from hydrogen ordeuterium.

In some embodiments, R⁶ is hydrogen. In some embodiments, R⁶ is hydrogenand at least one of R¹, R², R³, R⁴, R⁵, R⁷, or R⁸ is or containsdeuterium. In some embodiments, R⁶ is deuterium. In some embodiments, R⁶is deuterium and at least one of R¹, R², R³, R⁴, R⁵, R⁷, or R⁸ is orcontains deuterium.

As defined above and described herein, R⁷ is selected from hydrogen ordeuterium.

In some embodiments, R⁷ is hydrogen. In some embodiments, R⁷ is hydrogenand at least one of R¹, R², R³, R⁴, R⁵, or R⁸ is or contains deuterium.In some embodiments, R⁷ is deuterium. In some embodiments, R⁷ isdeuterium and at least one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is orcontains deuterium.

As defined above and described herein, R⁸ is selected from hydrogen ordeuterium.

In some embodiments, R⁸ is hydrogen. In some embodiments, R⁸ is hydrogenand at least one of R¹, R², R³, R⁴, R⁵, R or R⁷ is or containsdeuterium. In some embodiments, R⁸ is deuterium. In some embodiments, R⁸is deuterium and at least one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is orcontains deuterium.

In some embodiments, the present invention provides a compound offormulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein eachof R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is as defined above and described herein,and wherein each of R¹ and R² is as defined in an entry set forth inTable 1a, below.

TABLE 1a Entry R¹ R² i —NH₂ H ii —NH₂ D iii —NHD H iv —NHD D v —ND₂ H vi—ND₂ D

In some embodiments, the present invention provides a compound offormulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein eachof R¹, R², R⁵, R⁶, R⁷, and R⁸ is as defined above and described herein,and wherein each of R³ and R⁴ is as defined in an entry set forth inTable 1b, below.

TABLE 1b Entry R³ R⁴ i —CH₃ —CH₃ ii —CH₃ —CH₂D iii —CH₃ —CHD₂ iv —CH₃—CD₃ v —CH₂D —CH₃ vi —CH₂D —CH₂D vii —CH₂D —CHD₂ viii —CH₂D —CD₃ ix—CHD₂ —CH₃ x —CHD₂ —CH₂D xi —CHD₂ —CHD₂ xii —CHD₂ —CD₃ xiii —CD₃ —CH₃xiv —CD₃ —CH₂D xv —CD₃ —CHD₂ xvi —CD₃ —CD₃

In some embodiments, the present invention provides a compound offormulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein eachof R¹, R², R³, and R⁴ is as defined above and described herein, andwherein each of R⁵, R⁶, R⁷, and R⁸ is as defined in an entry set forthin Table 1c, below.

TABLE 1c Entry R⁵ R⁶ R⁷ R⁸ i H H H H ii H H H D iii H H D H iv H D H H vD H H H vi H H D D vii H D H D viii D H H D ix H D D H x D H D H xi D DH H xii H D D D xiii D H D D xiv D D H D xv D D D H xvi D D D D

In some embodiments, the present invention provides a compound offormulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein eachof R¹ and R² is as defined in an entry set forth in Table 1a, above,each of R³ and R⁴ is as defined in an entry set forth in Table 1b,above, and each of R⁵, R⁶, R⁷, and R⁸, is as defined in an entry setforth in Table 1c, above.

In some embodiments, the present invention provides a compound selectedfrom those recited in any of Table 1a, Table 1b, or Table 1c, or apharmaceutically acceptable salt thereof.

In some embodiments, present invention provides a compound of formula Iselected from these depicted in Table 2, below.

TABLE 2 Representative Compounds of Formula I

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

In some embodiments, the present invention provides a compound depictedin Table 2, above, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a deuterium-enrichedanalogue of a compound depicted in Table 2A, below, or apharmaceutically acceptable salt thereof, in which deuterium is enrichedat any available hydrogen.

TABLE 2A

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-40

I-41

I-42

I-43

I-44

I-45

I-46

I-47

In some embodiments, the present invention provides any compounddescribed herein comprising one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, or thirteen deuterium atoms.

In some embodiments, provided compounds comprise deuterium in an amountof about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 100%. As used herein in the context of deuterium enrichment,the term “about” means±2%.

In certain embodiments, the present invention provides any compounddescribed above and herein, or a pharmaceutically acceptable saltthereof.

In some embodiments, the present invention provides any compounddescribed above and herein in isolated form.

4. Uses of Compounds and Pharmaceutically Acceptable CompositionsThereof

Certain compounds described herein are found to be useful in scavengingtoxic aldehydes, such as MDA and HNE. The compounds described hereinundergo a Schiff base condensation with MDA, HNE, or other toxicaldehydes, and form a complex with the aldehydes in an energeticallyfavorable reaction, thus reducing or eliminating aldehydes available forreaction with a protein, lipid, carbohydrate, or DNA. Importantly,compounds described herein can react with aldehydes to form a compoundhaving a closed-ring structure that contains the aldehydes, thustrapping the aldehydes and preventing the aldehydes from being releasedback into the cellular milieu.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment is administered afterone or more symptoms have developed. In other embodiments, treatment isadministered in the absence of symptoms. For example, treatment isadministered to a susceptible individual prior to the onset of symptoms(e.g., in light of a history of symptoms and/or in light of genetic orother susceptibility factors). Treatment is also continued aftersymptoms have resolved, for example to prevent, delay or lessen theseverity of their recurrence.

The invention relates to compounds described herein for the treatment,prevention, and/or reduction of a risk of diseases, disorders, orconditions in which aldehyde toxicity is implicated in the pathogenesis.

Examples of the diseases, disorders, or conditions in which aldehydetoxicity is implicated include an ocular disease, disorder, orcondition, including, but not limited to, a corneal disease (e.g., dryeye syndrome, cataracts, keratoconus, bullous and other keratopathy, andFuch's endothelial dystrophy), other ocular disorders or conditions(e.g., allergic conjunctivitis, ocular cicatricial pemphigoid,conditions associated with PRK healing and other corneal healing, andconditions associated with tear lipid degradation or lacrimal glanddysfunction), and other ocular conditions associated with high aldehydelevels as a result of inflammation (e.g., uveitis, scleritis, ocularStevens Johnson Syndrome, ocular rosacea (with or without meibomiangland dysfunction)). In one example, the ocular disease, disorder, orcondition is not macular degeneration, such as age-related maculardegeneration (“AMD”), or Stargardt's disease. In a further example, theocular disease, disorder, or condition is dry eye syndrome, ocularrosacea, or uveitis.

Examples of the diseases, disorders, conditions, or indications in whichaldehyde toxicity is implicated also include non-ocular disorders,including psoriasis, topical (discoid) lupus, contact dermatitis, atopicdermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris,Sjogren-Larsson Syndrome and other ichthyosis, solar elastosis/wrinkles,skin tone firmness, puffiness, eczema, smoke or irritant induced skinchanges, dermal incision, a skin condition associated burn and/or wound,lupus, scleroderma, asthma, chronic obstructive pulmonary disease(COPD), rheumatoid arthritis, inflammatory bowel disease, sepsis,atherosclerosis, ischemic-reperfusion injury, Parkinson's disease,Alzheimer's disease, succinic semialdehyde dehydrogenase deficiency,multiple sclerosis, amyotrophic lateral sclerosis, diabetes, metabolicsyndrome, age-related disorders, and fibrotic diseases. In a furtherexample, the non-ocular disorder is a skin disease, disorder, orcondition selected from contact dermatitis, atopic dermatitis, allergicdermatitis, and. radiation dermatitis. In another example, thenon-ocular disorder is a skin disease, disorder, or condition selectedfrom Sjogren-Larsson Syndrome and a cosmetic indication associated burnand/or wound.

In a further example, the diseases, disorders, or conditions in whichaldehyde toxicity is implicated are an age-related disorder. Examples ofage-related diseases, disorders, or conditions include wrinkles,dryness, and pigmentation of the skin.

Examples of the diseases, disorders, or conditions in which aldehydetoxicity is implicated further include conditions associated with thetoxic effects of blister agents or burns from alkali agents. Thecompounds described herein reduce or eliminate toxic aldehydes and thustreat, prevent, and/or reduce a risk of these diseases or disorders.

In one embodiment, the invention relates to the treatment, prevention,and/or reduction of a risk of an ocular disease, disorder, or conditionin which aldehyde toxicity is implicated in the pathogenesis, comprisingadministering to a subject in need thereof a compound described herein.The ocular disease, disorder, or condition includes, but is not limitedto, a corneal disease (e.g., dry eye syndrome, cataracts, keratoconus,bullous and other keratopathy, and Fuch's endothelial dystrophy in thecornea), other ocular disorders or conditions (e.g., allergicconjunctivitis, ocular cicatricial pemphigoid, conditions associatedwith PRK healing and other corneal healing, and conditions associatedwith tear lipid degradation or lacrimal gland dysfunction), and otherocular conditions where inflammation leads to high aldehyde levels(e.g., uveitis, scleritis, ocular Stevens Johnson Syndrome, ocularrosacea (with or without meibomian gland dysfunction)). The oculardisease, disorder, or condition does not include macular degeneration,such as AMD, or Stargardt's disease. In one illustration, in the oculardisease, disorder, or condition, the amount or concentration of MDA orHNE is increased in the ocular tissues or cells. For example, the amountor concentration of aldehydes (e.g., MDA or HNE) is increased for atleast 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 5 fold, 10 fold as compared to that in normal ocular tissues orcells. Compounds described herein, such as Compound 9, decrease aldehyde(e.g., MDA and HNE) concentration in a time-dependent manner. The amountor concentration of aldehydes (e.g., MDA or HNE) can be measured bymethods or techniques known in the art, such as those described inTukozkan et al., Furat Tip Dergisi 11: 88-92 (2006).

In one class, the ocular disease, disorder, or condition is dry eyesyndrome. In a second class, the ocular disease, disorder, or conditionis a condition associated with PRK healing and other corneal healing.For example, the invention is directed to advancing PRK healing or othercorneal healing, comprising administering to a subject in need thereof acompound described herein. In a third class, the ocular disease,disorder, or condition is an ocular condition associated with highaldehyde levels as a result of inflammation (e.g., uveitis, scleritis,ocular Stevens Johnson Syndrome, and ocular rosacea (with or withoutmeibomian gland dysfunction). In a fourth class, the ocular disease,disorder, or condition is keratoconus, cataracts, bullous and otherkeratopathy, Fuchs' endothelial dystrophy, ocular cicatricialpemphigoid, or allergic conjunctivitis. The compound described hereinmay be administered topically or systemically, as described hereinbelow.

In a second embodiment, the invention relates to the treatment,prevention, and/or reduction of a risk of a skin disorder or conditionor a cosmetic indication, in which aldehyde toxicity is implicated inthe pathogenesis, comprising administering to a subject in need thereofa compound described herein. The skin disorder or condition includes,but is not limited to, psoriasis, scleroderma, topical (discoid) lupus,contact dermatitis, atopic dermatitis, allergic dermatitis, radiationdermatitis, acne vulgaris, and Sjogren-Larsson Syndrome and otherichthyosis, and the cosmetic indication is solar elastosis/wrinkles,skin tone firmness, puffiness, eczema, smoke or irritant induced skinchanges, dermal incision, or a skin condition associated burn and/orwound. In some embodiments, the invention related to age-relateddiseases, disorders, or conditions of the skin, as described herein.

Various skin disorders or conditions, such as atopic dermatitis, topical(discoid) lupus, psoriasis and scleroderma, are characterized by highMDA and HNE levels (Br J Dermatol 149: 248 (2003); JEADV 26: 833 (2012);Clin Rheumatol 25: 320 (2006)). In addition, ichthyosis characteristicof the Sjogren-Larsson Syndrome (SLS) originates from accumulation offatty aldehydes, which disrupts the normal function and secretion oflamellar bodies (LB) and leads to intercellular lipid deposits in theStrateum Corneum (SC) and a defective water barrier in the skin layer(W. B. Rizzo et al. (2010)). The enzyme, fatty aldehyde dehydrogenase,that metabolizes aldehydes is dysfunctional in SLS patients. Thus,compounds that reduce or eliminate aldehydes, such as the compoundsdescribed herein, can be used to treat, prevent, and/or reduction of arisk of skin disorders or conditions in which aldehyde toxicity isimplicated in the pathogenesis, such as those described herein.Furthermore, with an improvement to the water barrier and prevention ofaldehyde-mediated inflammation (including fibrosis and elastosis(Chairpotto et al. (2005)), many cosmetic indications, such as solarelastosis/wrinkles, skin tone, firmness (puffiness), eczema, smoke orirritant induced skin changes and dermal incision cosmesis, and skinconditions associated with burn and/or wound can be treated using themethod of the invention.

In one class, the skin disease, disorder, or condition is psoriasis,scleroderma, topical (discoid) lupus, contact dermatitis, atopicdermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris, orSjogren-Larsson Syndrome and other ichthyosis. In one exemplification,the skin disease, disorder, or condition is contact dermatitis, atopicdermatitis, allergic dermatitis, radiation dermatitis, orSjogren-Larsson Syndrome and other ichthyosis. In a second class, thecosmetic indication is solar elastosis/wrinkles, skin tone firmness,puffiness, eczema, smoke or irritant induced skin changes, dermalincision, or a skin condition associated burn and/or wound.

In a third embodiment, the invention relates to the treatment,prevention, and/or reduction of a risk of a condition associated withthe toxic effects of blister agents or burns from alkali agents in whichaldehyde toxicity is implicated in the pathogenesis, comprisingadministering to a subject in need thereof a compound described herein.

Blister agents include, but are not limited to, sulfur mustard, nitrogenmustard, and phosgene oxime. Toxic or injurious effects of blisteragents include pain, irritation, and/or tearing in the skin, eye, and/ormucous, and conjunctivitis and/or corneal damage to the eye. Sulfurmustard is the compound bis(2-chlorethyl) sulfide. Nitrogen mustardincludes the compounds bis(2-chlorethyl)ethylamine,bis(2-chlorethyl)methylamine, and tris(2-chlorethyl)amine. Sulfurmustard or its analogs can cause an increase in oxidative stress and inparticular in HNE levels, and by depleting the antioxidant defensesystem and thereby increasing lipid peroxidation, may induce anoxidative stress response and thus increase aldehyde levels (Jafari etal. (2010); Pal et al. (2009)). Antioxidants, such as Silibinin, whenapplied topically, attenuate skin injury induced from exposure to sulfurmustard or its analogs, and increased activities of antioxidant enzymesmay be a compensatory response to reactive oxygen species generated bythe sulfur mustard (Jafari et al. (2010); Tewari-Singh et al. (2012)).Further, intervention to reduce free radical species was an effectivetreatment post exposure for phosgene induced lung injury (Sciuto et al.(2004)). Thus, compounds that reduce or eliminate aldehydes, such ascompounds described herein, can be used to treat, prevent, and/or reducea risk of a condition associated with the toxic effects of blisteragents, such as sulfur mustard, nitrogen mustard, and phosgene oxime.

Alkali agents include, but are not limited to, lime, lye, ammonia, anddrain cleaners. Compounds that reduce or eliminate aldehydes, such ascompounds described herein, can be used to treat, prevent, and/or reducea risk of a condition associated with burns from an alkali agent.

In a fourth embodiment, the invention relates to the treatment,prevention, and/or reduction of a risk of an autoimmune,immune-mediated, inflammatory, cardiovascular, or neurological disease,disorder, or condition, or metabolic syndrome, or diabetes, in whichaldehyde toxicity is implicated in the pathogenesis, comprisingadministering to a subject in need thereof a compound described herein.The autoimmune or immune-mediated disease, disorder, or conditionincludes, but is not limited to, lupus, scleroderma, asthma, chronicobstructive pulmonary disease (COPD), and rheumatoid arthritis. Theinflammatory disease, disorder, or condition includes, but is notlimited to, rheumatoid arthritis, inflammatory bowel disease (e.g.,Crohn's disease and ulcerative colitis), sepsis, and fibrosis (e.g.,renal, hepatic, pulmonary, and cardiac fibrosis). The cardiovasculardisease, disorder, or condition includes, but is not limited to,atherosclerosis and ischemic-reperfusion injury. The neurologicaldisease, disorder, or condition includes, but is not limited to,Parkinson's disease, Alzheimer's disease, succinic semialdehydedehydrogenase deficiency, multiple sclerosis, amyotrophic lateralsclerosis, and the neurological aspects of Sjogren-Larsson Syndrome(cognitive delay and spasticity).

A skilled person would understand that the disease, disorder, orcondition listed herein may involve more than one pathologicalmechanism. For example, a disease, disorder, or condition listed hereinmay involve dysregulation in the immunological response and inflammatoryresponse. Thus, the above categorization of a disease, disorder, orcondition is not absolute, and the disease, disorder, or condition maybe considered an immunological, an inflammatory, a cardiovascular, aneurological, and/or metabolic disease, disorder, or condition.

Individuals with deficiencies in aldehyde dehydrogenase are found tohave high aldehyde levels and increased risk of Parkinson's disease(PNAS 110:636 (2013)) and Alzheimer's disease (BioChem Biophys ResCommun. 273:192 (2000)). In Parkinson's disease, aldehydes specificallyinterfere with dopamine physiology (Free Radic Biol Med, 51: 1302(2011); Mol Aspects Med, 24: 293 (2003); Brain Res, 1145: 150 (2007)).In addition, aldehydes levels are elevated in multiple sclerosis,amyotrophic lateral sclerosis, autoimmune diseases such as lupus,rheumatoid arthritis, lupus, psoriasis, scleroderma, and fibroticdiseases, and increased levels of HNE and MDA are implicated in theprogression of atherosclerosis and diabetes (J. Cell. Mol. Med., 15:1339 (2011); Arthritis Rheum 62: 2064 (2010); Clin Exp Immunol, 101: 233(1995); Int J Rheum Dis, 14: 325 (2011); JEADV 26: 833 (2012); ClinRheumatol 25: 320 (2006); Gut 54: 987 (2005); J Am Soc Nephrol 20: 2119(2009)). MDA is further implicated in the increased formation of foamcells leading to atherosclerosis (Leibundgut et al., Current Opinion inPharmacology 13: 168 (2013)). Also, aldehyde-related toxicity plays animportant role in the pathogenesis of many inflammatory lung diseases,such as asthma and chronic obstructive pulmonary disease (COPD) (Bartoliet al., Mediators of Inflammation 2011, Article 891752). Thus, compoundsthat reduce or eliminate aldehydes, such as compounds described herein,can be used to treat, prevent, and/or reduce a risk of an autoimmune,immune-mediated, inflammatory, cardiovascular, or neurological disease,disorder, or condition, or metabolic syndrome, or diabetes. For example,compounds described herein prevent aldehyde-mediated cell death inneurons. Further, compounds described herein downregulate a broadspectrum of pro-inflammatory cytokines and/or upregulateanti-inflammatory cytokines, which indicates that compounds describedherein are useful in treating inflammatory diseases, such as multiplesclerosis and amyotrophic lateral sclerosis.

As discussed above, a disclosed composition may be administered to asubject in order to treat or prevent macular degeneration and otherforms of retinal disease whose etiology involves the accumulation of A2Eand/or lipofuscin. Other diseases, disorders, or conditionscharacterized by the accumulation A2E may be similarly treated.

In one embodiment, a compound is administered to a subject that reducesthe formation of A2E. For example, the compound may compete with PE forreaction with trans-RAL, thereby reducing the amount of A2E formed. Inanother embodiment, a compound is administered to a subject thatprevents the accumulation of A2E. For example, the compound competes sosuccessfully with PE for reaction with trans-RAL, no A2E is formed.

Individuals to be treated fall into three groups: (1) those who areclinically diagnosed with macular degeneration or other forms of retinaldisease whose etiology involves the accumulation of A2E and/orlipofuscin on the basis of visual deficits (including but not limited todark adaptation, contrast sensitivity and acuity) as determined byvisual examination and/or electroretinography, and/or retinal health asindicated by fundoscopic examination of retinal and RPE tissue fordrusen accumulations, tissue atrophy and/or lipofuscin fluorescence; (2)those who are pre-symptomatic for macular degenerative disease butthought to be at risk based on abnormal results in any or all of thesame measures; and (3) those who are pre-symptomatic but thought to beat risk genetically based on family history of macular degenerativedisease and/or genotyping results showing one or more alleles orpolymorphisms associated with the disease. The compositions areadministered topically or systemically at one or more times per month,week or day. Dosages may be selected to avoid side effects, if any, onvisual performance in dark adaptation. Treatment is continued for aperiod of at least one, three, six, or twelve or more months. Patientsmay be tested at one, three, six, or twelve months or longer intervalsto assess safety and efficacy. Efficacy is measured by examination ofvisual performance and retinal health as described above.

In one embodiment, a subject is diagnosed as having symptoms of maculardegeneration, and then a disclosed compound is administered. In anotherembodiment, a subject may be identified as being at risk for developingmacular degeneration (risk factors include a history of smoking, age,female gender, and family history), and then a disclosed compound isadministered. In another embodiment, a subject may have dry AMD in botheye, and then a disclosed compound is administered. In anotherembodiment, a subject may have wet AMD in one eye but dry AMD in theother eye, and then a disclosed compound is administered. In yet anotherembodiment, a subject may be diagnosed as having Stargardt disease andthen a disclosed compound is administered. In another embodiment, asubject is diagnosed as having symptoms of other forms of retinaldisease whose etiology involves the accumulation of A2E and/orlipofuscin, and then the compound is administered. In another embodimenta subject may be identified as being at risk for developing other formsof retinal disease whose etiology involves the accumulation of A2Eand/or lipofuscin, and then the disclosed compound is administered. Insome embodiments, a compound is administered prophylactically. In someembodiments, a subject has been diagnosed as having the disease beforeretinal damage is apparent. For example, a subject is found to carry agene mutation for ABCA4 and is diagnosed as being at risk for Stargardtdisease before any ophthalmologic signs are manifest, or a subject isfound to have early macular changes indicative of macular degenerationbefore the subject is aware of any effect on vision. In someembodiments, a human subject may know that he or she is in need of themacular generation treatment or prevention.

In some embodiments, a subject may be monitored for the extent ofmacular degeneration. A subject may be monitored in a variety of ways,such as by eye examination, dilated eye examination, fundoscopicexamination, visual acuity test, and/or biopsy. Monitoring can beperformed at a variety of times. For example, a subject may be monitoredafter a compound is administered. The monitoring can occur, for example,one day, one week, two weeks, one month, two months, six months, oneyear, two years, five years, or any other time period after the firstadministration of a compound. A subject can be repeatedly monitored. Insome embodiments, the dose of a compound may be altered in response tomonitoring.

In some embodiments, the disclosed methods may be combined with othermethods for treating or preventing macular degeneration or other formsof retinal disease whose etiology involves the accumulation of A2Eand/or lipofuscin, such as photodynamic therapy. For example, a patientmay be treated with more than one therapy for one or more diseases ordisorders. For example, a patient may have one eye afflicted with dryform AMD, which is treated with a compound of the invention, and theother eye afflicted with wet form AMD which is treated with, e.g.,photodynamic therapy.

In some embodiments, a compound for treating or preventing maculardegeneration or other forms of retinal disease whose etiology involvesthe accumulation of A2E and/or lipofuscin may be administeredchronically. The compound may be administered daily, more than oncedaily, twice a week, three times a week, weekly, biweekly, monthly,bimonthly, semiannually, annually, and/or biannually.

Sphingosine 1-phosphate, a bioactive signaling molecule with diversecellular functions, is irreversibly degraded by the endoplasmicreticulum enzyme sphingosine 1-phosphate lyase, generatingtrans-2-hexadecenal and phosphoethanolamine. It has been demonstratedthat trans-2-hexadecenal causes cytoskeletal reorganization, detachment,and apoptosis in multiple cell types via a JNK-dependent pathway. SeeBiochem Biophys Res Commun. 2012 Jul. 20; 424(1):18-21. These findingsand the known chemistry of related α,β-unsaturated aldehydes raise thepossibility that trans-2-hexadecenal interact with additional cellularcomponents. It was shown that it reacts readily with deoxyguanosine andDNA to produce the diastereomeric cyclic 1,N(2)-deoxyguanosine adducts3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-tridecylpyrimido[1,2-a]purine-10(3H)oneand3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one.These findings demonstrate that trans-2-hexadecenal producedendogenously by sphingosine 1-phosphate lyase react directly with DNAforming aldehyde-derived DNA adducts with potentially mutagenicconsequences.

Succinic semialdehyde dehydrogenase deficiency (SSADHD), also known as4-hydroxybutyric aciduria or gamma-hydroxybutyric aciduria, is the mostprevalent autosomal-recessively inherited disorder of GABA metabolism(Vogel et al. 2013), manifests a phenotype of developmental delay andhypotonia in early childhood, and severe expressive language impairmentand obsessive-compulsive disorder in adolescence and adulthood. Epilepsyoccurs in half of patients, usually as generalized tonic-clonic seizuresalthough sometimes absence and myoclonic seizures occur (Pearl et al.2014). Greater than two-thirds of patients manifest neuropsychiatricproblems (i.e., ADHD, OCD and aggression) in adolescence and adulthood,which can be disabling. Metabolically, there is accumulation of themajor inhibitory neurotransmitter GABA and gamma-hydroxybutyrate (GHB),a neuromodulatory monocarboxylic acid (Snead and Gibson 2005). Inaddition, several other intermediates specific to this disorder havebeen detected both in patients and the corresponding murine model.Vigabatrin (VGB; γ-vinylGABA), an irreversible inhibitor ofGABA-transaminase, is a logical choice for treatment of SSADH deficiencybecause it will prevent the conversion of GABA to GHB. Outcomes havebeen mixed, and in selected patients treatment has led to deterioration(Good 2011; Pellock 2011; Escalera et al. 2010; Casarano et al. 2011;Matern et al. 1996; A1-Essa et al. 2000). Targeted therapy for SSADHdeficiency remains elusive and interventions palliative.

5. Pharmaceutically Acceptable Compositions

The compounds and compositions, according to the method of the presentinvention, are administered using any amount and any route ofadministration effective for treating or lessening the severity of adisorder provided above. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particular agent, itsmode of administration, and the like. Compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts.

Pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention are administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The compounds of the invention can also be administered topically, suchas directly to the eye, e.g., as an eye-drop or ophthalmic ointment. Eyedrops typically comprise an effective amount of at least one compound ofthe invention and a carrier capable of being safely applied to an eye.For example, the eye drops are in the form of an isotonic solution, andthe pH of the solution is adjusted so that there is no irritation of theeye. In many instances, the epithelial barrier interferes withpenetration of molecules into the eye. Thus, most currently usedophthalmic drugs are supplemented with some form of penetrationenhancer. These penetration enhancers work by loosening the tightjunctions of the most superior epithelial cells (Burstein, 1985, TransOphthalmol Soc U K 104(Pt 4): 402-9; Ashton et al., 1991, J PharmacolExp Ther 259(2): 719-24; Green et al., 1971, Am J Ophthalmol 72(5):897-905). The most commonly used penetration enhancer is benzalkoniumchloride (Tang et al., 1994, J Pharm Sci 83(1): 85-90; Burstein et al,1980, Invest Ophthalmol Vis Sci 19(3): 308-13), which also works aspreservative against microbial contamination. It is typically added to afinal concentration of 0.01-0.05%.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

All features of each of the aspects of the invention apply to all otheraspects mutatis mutandis.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Example 1. General Reaction Sequence for Compounds

Deuterium-labeled aldehyde trapping agents were made as described inU.S. patent application publication US 2013/0190500, published Jul. 23,2013, optionally using deuterium-labeled intermediates at the sitesindicated in Scheme 1. Exemplary methods are described further below.

Example 2: Synthesis of A-1

1-(3-ethoxy-2,3-dioxopropyl)pyridin-1-ium bromide. To a 2 L round bottomflask was charged ethanol (220 mL) and pyridine (31 g, 392 mmol), andthe resulting solution was stirred at a moderate rate of agitation undernitrogen. To this solution was added ethyl bromopyruvate (76.6 g, 354mmol) in a slow, steady stream. The reaction mixture was allowed to stirat 65±5° C. for 2 hours.

Example 3: Synthesis of A-2a

1-(6-chloro-2-(ethoxycarbonyl)quinolin-3-yl)pyridin-1-ium bromide. Uponcompletion of the 2 hour stir time in Example 2, the reaction mixturewas slowly cooled to 18-22° C. The flask was vacuum-purged three timesat which time 2-amino-5-chloro-benzaldehyde (ACB) (50.0 g, 321 mmol) wasadded directly to the reaction flask as a solid using a long plasticfunnel. Pyridine (64.0 g, 809 mmol) was added followed by an EtOH rinse(10 mL) and the reaction mixture was heated at 80±3° C. under nitrogenfor about 16 hours (overnight) at which time HPLC analysis indicatedthat the reaction was effectively complete.

Example 4: Synthesis of A-2b

1-(6-chloro-2-(ethoxycarbonyl)quinolin-3-yl-4-d)pyridin-1-ium. CompoundA-2b is prepared in a manner similar to A-2a (See Example 3),substituting 2-amino-5-chloro-benzaldehyde (ACB) for2-amino-5-chloro-benzaldehyde-d.

Example 5: Synthesis of A-3a

Ethyl 3-amino-6-chloroquinoline-2-carboxylate. The reaction mixture fromExample 3 was cooled to about 70° C. and morpholine (76.0 g, 873 mmol))was added to the 2 L reaction flask using an addition funnel. Thereaction mixture was heated at 80±2° C. for about 2.5 hours at whichtime the reaction was considered complete by HPLC analysis (area % ofA-3a stops increasing). The reaction mixture was cooled to 10-15° C. forthe quench, work up, and isolation.

To the 2 L reaction flask was charged water (600 g) using the additionfunnel over 30-60 minutes, keeping the temperature below 15° C. byadjusting the rate of addition and using a cooling bath. The reactionmixture was stirred for an additional 45 minutes at 10-15° C. then thecrude A-3a was isolated by filtration using a Buchner funnel. The cakewas washed with water (100 mL×4) each time allowing the water topercolate through the cake before applying a vacuum. The cake was airdried to provide crude A-3a as a nearly dry brown solid. The cake wasreturned to the 2 L reaction flask and heptane (350 mL) and EtOH (170mL) were added, and the mixture heated to 70±3° C. for 30-60 minutes.The slurry was cooled to 0-5° C. and isolated by filtration undervacuum. The A-3a was dried in a vacuum drying oven under vacuum and35±3° C. overnight (16-18 hours) to provide A-3a as a dark green solid.

Example 6: Synthesis of A-3b

Ethyl 3-amino-6-chloroquinoline-2-carboxylate-4-d. Compound A3-b isprepared in a similar manner as compound A3-a (See Example 5),substituting the reaction mixture of A2-a for that of A2-b.

Example 7: Synthesis of NS2

2-(3-amino-6-chloroquinolin-2-yl)propan-2-ol. To a 2 L round bottomflask was charged methylmagnesium chloride (200 mL of 3.0 M solution inTHF, 600 mmol). The solution was cooled to 0-5° C. using an ice bath.

A 500 mL flask (magnetic stirring) was charged with 22.8 grams A-3a fromExample 5 and THF (365 mL), stirred to dissolve, and then transferred toan addition funnel on the 2 L Reaction Flask. The A-3a solution wasadded drop-wise to the reaction flask over 5.75 hours, keeping thetemperature of the reaction flask between 0-5° C. throughout theaddition. At the end of the addition the contents of the flask werestirred for an additional 15 minutes at 0-5° C. then the cooling bathwas removed and the reaction was allowed to stir overnight at ambienttemperature.

The flask was cooled in an ice bath and the reaction mixture wascarefully quenched by adding EtOH (39.5 g, 857 mmol) drop-wise to thereaction mixture, keeping the temperature of the reaction mixture below15° C. during the course of the addition. An aqueous solution of NH₄Cl(84.7 g NH₄Cl in 415 mL water) was then carefully added and the mixturestirred under moderate agitation for about 30 minutes then transferredto a separatory funnel to allow the layers to separate. Solids werepresent in the aqueous phase so HOAc (12.5 g) was added and the contentsswirled gently to obtain a nearly homogeneous lower aqueous phase. Thelower aqueous layer was transferred back to the 2 L reaction flask andstirred under moderate agitation with 2-methylTHF (50 mL) for about 15minutes. The original upper organic layer was reduced in volume toapproximately 40 mL using a rotary evaporator at <40° C. and vacuum asneeded. The phases in the separatory funnel were separated and the upper2-MeTHF phase combined with the product residue, transferred to a 500 mLflask, and vacuum distilled to an approximate volume of 25 mL. To thisresidue was added 2-MeTHF (50 mL) and distilled to an approximate volumeof 50 mL. The crude compound NS2 solution was diluted with 2-MeTHF (125mL), cooled to 5-10° C., and 2M H₂SO₄ (aq) (250 mL) was slowly added andthe mixture stirred for 30 minutes as the temperature was allowed toreturn to ambient. Heptane (40 mL) was charged and the reaction mixturestirred for an additional 15 minutes then transferred to a separatoryfunnel, and the layers were allowed to separate. The lower aqueousproduct layer was extracted with additional heptane (35 mL), then thelower aqueous phase was transferred to a 1 L reaction flask equippedwith a mechanical stirrer, and the mixture was cooled to 5-10° C. Thecombined organic layers were discarded. A solution of 25% NaOH (aq) wasprepared (NaOH, 47 g, water, 200 mL) and slowly added to the 1 Lreaction flask to bring the pH to a range of 6.5-8.5.

EtOAc (250 mL) was added and the mixture was stirred overnight. Themixture was transferred to a separatory funnel and the lower phasediscarded. The upper organic layer was washed with brine (25 mL), thenthe upper organic product layer was reduced in volume on a rotaryevaporator to obtain a obtain the crude compound NS2 as a dark oil thatsolidified within a few minutes. The crude compound NS2 was dissolved inEtOAc (20 mL) and filtered through a plug of silica gel (23 g) elutingwith 3/1 heptane/EtOAc until all compound NS2 was eluted (approximately420 mL required) to remove most of the dark color of compound NS2. Thesolvent was removed in vacuo to provide 14.7 g of compound NS2 as a tansolid. Compound NS2 was taken up in EtOAc (25 mL) and eluted through acolumn of silica gel (72 g) using a mobile phase gradient of 7/1heptane/EtOAc to 3/1heptane/EtOAc (1400 mL total). The solvent fractionscontaining compound NS2 were stripped. Compound NS2 was diluted withEtOAc (120 mL) and stirred in a flask with Darco G-60 decolorizingcarbon (4.0 g) for about 1 hour. The mixture was filtered through celiteusing a firtted funnel, rinsing the cake with EtOAc (3×15 mL). Thecombined filtrates were stripped on a rotary evaporator and compound NS2dissolved in heptane (160 mL)/EtOAc(16 mL) at 76° C. The homogeneoussolution was slowly cooled to 0-5° C., held for 2 hours, then compoundNS2 was isolated by filtration. After drying in a vacuum oven for 5hours at 35° C. under best vacuum, compound NS2 was obtained as a whitesolid. HPLC purity: 100% (AUC); HPLC (using standard conditions): A-2:7.2 minutes; A-3: 11.6 minutes.

Example 8: Synthesis of I-1

2-(6-chloroquinolin-2-yl)propan-1,1,1,3,3,3-d₆-2-ol. Compound I-1 wasprepared in a similar manner to compound NS2 (See Example 7),substituting methylmagnesium chloride with methyl-d3-magnesisum iodide(99 atom % D). The reaction of A3-a with 5.3 mol equiv 1.0 Mmethyl-d₃-magnesium iodide (99 atom % D) in ether/THF gave a 20% yieldof I-1. MS (ESI): m/z 242.9 (M+1); ¹H NMR (CDCl₃, 300 MHz) δ: 7.80 (d,J=6 Hz, 1H), 7.51 (d, J=2 Hz, 1H), 7.33 (dd, J=2 and 2 Hz, 1H), 7.07 (s,1H), 4.68 (br s, 2H), 3.83 (br s, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ: 154.3,139.5, 139.1, 132.4, 130.6, 130.0, 126.3, 123.7, 116.4, 76.8, 74.9.

Example 9: Synthesis of I-2

2-(6-chloroquinolin-2-yl-4-d)propan-2-ol. Compound 1-2 is prepared in asimilar manner to compound NS2 (See Example 7), substituting A3-a forA3-b.

Preparation of ACB

After a N₂ atmosphere had been established and a slight stream of N₂ wasflowing through the vessel, platinum, sulfided, 5 wt % on carbon,reduced, dry (9.04 g, 3.0 wt % vs the nitro substrate) was added to a 5L heavy walled pressure vessel equipped with a large magnetic stir-barand a thermocouple. MeOH (1.50 L), 5-chloro-2-nitrobenzaldehyde (302.1g, 1.63 mol), further MeOH (1.50 L) and Na₂CO₃ (2.42 g, 22.8 mmol, 0.014equiv) were added. The flask was sealed and stirring was initiated at450 rpm. The solution was evacuated and repressurized with N₂ (35 psi),2×. The flask was evacuated and repressurized with H₂ to 35 psi. Thetemperature of the solution reached 30° C. w/in 20 min. The solution wasthen cooled with a water bath. Ice was added to the water bath tomaintain a temperature below 35° C. Every 2 h, the reaction wasmonitored by evacuating and repressurizing with N₂ (5 psi), 2× prior toopening. The progress of the reaction could be followed by TLC:5-Chloro-2-nitrobenzaldehyde (R_(f)=0.60, CH₂Cl₂, UV) and theintermediates (R_(f)=0.51, CH₂Cl₂, UV and R_(f)=0.14, CH₂Cl₂, UV) wereconsumed to give ACB (R_(f)=0.43, CH₂Cl₂, UV). At 5h, the reaction hadgone to 98% completion (GC), and was considered complete. To a 3 Lmedium fritted funnel was added celite (ca. 80 g). This was settled withMeOH (ca. 200 mL) and pulled dry with vacuum. The reduced solution wastransferred via cannula into the funnel while gentle vacuum was used topull the solution through the celite plug. This was chased with MeOH(150 mL 4×). The solution was transferred to a 5 L three-neckedround-bottom flask. At 30° C. on a rotavap, solvent (ca. 2 L) wasremoved under reduced pressure. An N₂ blanket was applied. The solutionwas transferred to a 5 L four-necked round-bottomed flask equipped withmechanical stirring and an addition funnel. Water (2.5 L) was addeddropwise into the vigorously stirring solution over 4 h. The slurry wasfiltered with a minimal amount of vacuum. The collected solid was washedwith water (1.5 L 2×), 2-propanol (160 mL) then hexanes (450 mL 2×). Thecollected solid (a canary yellow, granular solid) was transferred to a150×75 recrystallizing dish. The solid was then dried under reducedpressure (26-28 in Hg) at 40° C. overnight in a vacuum-oven. ACB (>99A %by HPLC) was stored under a N₂ atmosphere at 5° C.

Example 10: In Vitro Assays LDH Cytotoxicity Assay

Primary rat cortical cultures are placed in an incubator for 24 or 48hours and treated with various concentrations of disclosed compounds.Then 20 μL of the culture media is removed for an LDH assay as describedin Bergmeyer et al., Methods of Enzymatic Analysis, 3^(rd) ed. (1983).

ELISA Assay to Determine Amount of Circulating Cytokines

Male C57BI/6 mice are dosed with disclosed compounds 30 minutes beforethey were exposed to LPS (20 mg/kg). Two hours after the LPS exposure,blood is collected from the mice and an ELISA is conducted to determinethe amount of circulating cytokines. Treatment with disclosed compoundsleads to reduction in proinflammatory cytokines, such as IL-5 and IL-1β,IL-17, and TNF. Also, treatment with disclosed compounds results inelevated anti-inflammatory cytokines, such as IL-10. In addition,various other chemokines, such as eotaxin, IL-12, IP-10, LIF, MCP-1,MIG, MIP, and RANTES, are also decreased by treatment with disclosedcompounds.

Assay to Evaluate Efficacy in Treating Contact Dermatitis

To determine the efficacy of the disclosed compounds in treating contactdermatitis, phorbol myristate acetate (“PMA”) is applied topically (2.5μg in 20 μL) to both the anterior and posterior portions of the rightpinna of mice (N=10 per group). As a control, the left pinna receives 20μL of ethanol (PMA excipient) to both the anterior and posteriorportions. Six hours after the PMA application, both the right and leftpinna thickness is determined. Measurements are determined at leasttwice from the same region of both ears, with care taken not to includehair or folded pinna.

Assay to Evaluate the Efficacy in Treating Allergic Dermatitis

To measure the efficacy of the disclosed compounds in treating allergicdermatitis, oxazolone (“OXL”) is applied (1.5%, 100 μL in acetone) tothe shaved abdomens of mice. Seven days later, the thickness of thepinna of the OXL treated mice is determined. Then the disclosedcompounds (100 mg/kg) or the vehicle (i.e., Captisol) is administeredintraperitoneally to mice followed by topical application of OXL (1%, 20μL) 30 min later to both the anterior and posterior portions of theright pinna. As a control, the left pinna receives 20 μL of acetone (OXLexcipient) to both the anterior and posterior portions. The thickness ofthe pinna of both ears is measured again 24 hours later. N=10 per group.

Assay to Measure Aldehyde Trapping

To separate reaction vials is added each disclosed compound, (0.064mmol), MDA salt (22.7% MDA, 0.064 mmol), and glyceryl trioleate (600mg). To the mixture is added 20 wt % Capitsol in aqueous PBS (˜2.5 ml),followed by linoleic acid (600 mg). The reaction mixture is stirredvigorously at ambient temperature and monitored by LC/MS. The disclosedcompounds quickly react with MDA to form MDA adducts.

Schiff Base Confirmation

UV/VIS spectroscopy is used to monitor Schiff base condensation of RALwith the primary amine of a compound of the invention. The in vitroanalysis of the Schiff base condensation product with RAL is performedfor the disclosed compounds.

In the solution phase analysis, the λ_(max) value of both the freecompound and the RAL Schiff base condensation product (RAL-SBC) aremeasured along with the value for tau of the RAL-SBC. As used herein,“RAL-SBC” means the Schiff base condensation product of RAL and aRAL-compound. Solution phase analysis is performed using a 100:1 mixtureof compound and RAL using protocols known in the art. Several solventsystems were tested including aqueous, ethanol, octanol, andchloroform:methanol (various e.g., 2:1). The solution kinetics aremeasured and found to be highly dependent on solvent conditions.

Solid phase analysis of the Schiff base condensation is also performedusing a 1:1 mixture of compound to RAL. The solid phase analysis isperformed using protocols known in the art. The mixture is dried undernitrogen and condensation reaction occurs to completion.

Lipid phase analysis is performed using protocols known in the art andλ_(max), tau (RAL-SBC vs. APE/A2PE), and competitive inhibition aremeasured. Liposome conditions are closer to in situ conditions.

ERG Analysis of Dark Adaptation

Dark adaptation is the recovery of visual sensitivity following exposureto light. Dark adaptation has multiple components including both fast(neuronal) processes and a slow (photochemical) process. Regeneration ofvisual pigment is related to the slow photochemical process. Darkadaptation rates are measured for several reasons. Night blindnessresults from a failure to dark adapt (loss of visual light sensitivity).It is possible to find a safe dose for night vision by measuring drugeffects on dark adapted visual light sensitivity.

An electroretinogram (ERG) is used to measure dark adaptation undernormal vs. drug conditions. ERG is the measurement of the electric fieldpotential emitted by retinal neurons during their response to anexperimentally defined light stimulus. More specifically, ERG measuresretinal field potentials at the cornea after a flash of light (e.g., 50ms). Field strengths are 102 to 103 microvolts, originating in retinalcells.

ERG is a non-invasive measurement which can be performed on eitherliving subjects (human or animal) or a hemisected eye in solution thathas been removed surgically from a living animal. ERG requires generalanesthesia which slows dark adaptation and must be factored intoexperimental design.

In a typical ERG analysis of dark adaptation experiment, every rat isdark adapted for hours to reach a consistent state of light sensitivity.The rat is then “photo-bleached,” i.e., exposed briefly to light strongenough to transiently deplete the retina of free 11-cis-RAL (e.g., 2 minat 300 lux). The rat is then returned to dark immediately to initiatedark adaptation, i.e., recovery of light sensitivity due to regenerationof visual pigment. ERG is used to measure how quickly the rat adapts todark and recovers light sensitivity. Specifically, a criterion responsevariable is defined for light sensitivity.

The ERG measurement is taken after a specific duration of post-bleachdark recovery (e.g., 30 min) determined previously by kinetic analysis.A curve fit is used to calculate value for the sensitivity variable andshows recovery with anesthesia in the same rat including dark adaptationkinetics for Y₅₀ and σ. Slower adaptation is observed with less lightsensitivity where Y₅₀ reaches −4.0 and tau=22.6 min. Faster adaptationis observed with more light sensitivity where Y₅₀ reaches −5.5 andtau=9.2 min.

The same paradigm as described above is followed for dose ranging. Inthe ERG dose ranging protocol, compounds i.p. lowers light sensitivityof dark adapted rats in a dose dependent manner. The effect on visiondecreases after 3 hours.

NMR Analysis of RAL Reaction

NMR spectroscopy is used to monitor Schiff base condensation and ringformation of RAL with the primary amine of a compound of the invention.

Inhibition of A2E Formation

This experiment is designed to establish proof of concept that chronici.p. injection of a RAL-trap compound lowers the accumulation rate ofA2E in wild type Sprague Dawley rats. These experiments compare thetreatment efficacy of RAL-trap compounds to that of control compoundsand lack of treatment.

Materials and Methods:

The study is performed with wild type Sprague Dawley rats. Rat treatmentgroups include, for example, 8 rats of mixed gender per treatmentcondition. Each animal is treated with one of the following conditions:

-   -   Controls: (1) 13-cis retinoic acid to inhibit retinoid binding        sites of visual cycle proteins as a protocol control, in that        such treatment reduces the amount of free trans-RAL that is        released and thereby available to form A2E, but with undesirable        side effects of night blindness, and (2) a commercially        available compound known clinically to modulate retinal function        in humans and known experimentally to form a Schiff base adduct        with free RAL, both in vitro and in vivo in animal models.    -   Vehicle    -   Compound    -   Untreated

The disclosed compounds are tested across a dose range including 1, 5,15, and 50 mg/kg. Treatment is administered daily for 8 weeks by i.p.injection.

Chemistry:

The experiments use a variety of chemistry services. For example, theseexperiments use commercially available compounds with analyticalspecification sheets to characterize the impurities. Compounds are alsosynthesized. Compounds are prepared in quantities sufficient for therequired dosing. Formulations of the compound are suitable for use ininitial animal safety studies involving intraperitoneal (i.p.)injection. The following three attributes of the Schiff base reactionproduct of trans-RAL with compounds of the invention are determined:

-   -   stability with respect to reaction rates    -   absorption properties, specifically uv-vis absorption maxima and        extinction coefficients (see e.g., FIG. 5 in Rapp and Basinger,        Vision Res. 22:1097, 1982) or NMR spectral analysis of reaction        kinetics    -   log P and log D solubility values e.g. calculated

Biology and Biochemistry:

The experiments described herein use a variety of biology andbiochemistry services. A “no effect level” (NOEL) dose of compounds ofthe invention for daily treatment with an eye drop formation isestablished, e.g., in the rabbit with an ocular irritation protocol andin the rodent with ERG measurement of dark adaptation in visualresponses to light stimulation. After treatment and before eyeenucleation, the following non-invasive assays are performed in animals,e.g., rabbits:

-   -   RPE and photoreceptor cell degeneration, as evident by fundus        photography (Karan, et al. 2005, PNAS 102:4164)    -   Extracellular drusen and intracellular lipofuscin as measured by        fundus fluorescent photography (Karan et al. 2005)

Light responses are characterized by ERG (Weng, et al., Cell 98:13,1999). Intracellular A2E concentration of retinal RPE cell extracts ismeasured in all treated animals upon the conclusion of the treatmentprotocol using an analytical method such as those described by Karan etal., 2005; Radu et al., 2003; and Parish et al., PNAS 95:14609, 1998.For example, in a sample of treated animals, one eye is assayed, and theother eye is saved for histology analysis (as described below). In theremaining animals, both eyes are assayed separately for A2E formation.

In the post-treatment eyes set aside for histology (as described above),the morphology of retinal and RPE tissue is assessed with lightmicroscopy histology techniques (Karan et al. 2005, with the exceptionthat electron microscopy is not used in the experiments describedherein).

The safety of the treatment regimen is assessed for example using acombination of:

-   -   Daily documented observation of animal behavior and feeding        habits throughout the treatment period    -   Visual performance as measured by ERG at the end of the        treatment period    -   Ocular histology at the end of the treatment

Example 11: Cross-Species Metabolite Profiling Test of Deuterated NS2(NS2-D6; Compound I-1) Purpose:

Once administered to an animal, small molecules may undergo a variety ofreactions to produce an array of metabolites. The exact metabolitesproduced depend on many factors such as the animal, the molecularstructure, and the tissue distribution of the molecule. Metabolism ofsmall molecules may serve the purpose of increasing water solubility toaid in excretion of the molecule in the urine or feces, or may simply bethe result of adventitious enzyme-catalyzed reactions. Exemplarymetabolites include oxidation and glucuronidation products. It is oftenimpossible to predict the distribution and amount of metabolitesproduced. One purpose of this study was to conduct a cross speciesmetabolite profiling of test article NS2 in cryopreserved primaryhepatocytes. As described below, a variety of NS2 metabolites wereproduced and the distribution of metabolites varied significantly acrossdifferent species. It is often desirable to decrease the number andamount of metabolites of small molecule drugs, for example to increasedrug half-life in the body and/or prevent conversion to toxic orinactive metabolites. With this knowledge in hand, deuteration of NS2was explored as a possible route to decrease the number and amount ofmetabolites. Deuteration of a molecule (i.e., replacement of one or morehydrogen atoms with deuterium) often has significant effects on therates of production of metabolites; however, the effects of deuterationon a given molecule are almost impossible to predict in most cases.Therefore, metabolite profiling was performed for deuterated-NS2 inhuman hepatocytes.

Study Conditions:

This study was performed under non-GLP conditions. All work wasperformed with appropriate local health regulations and ethicalapproval.

Experimental Design: Sample Analysis

Samples were analyzed by LC-MS/MS using a SCIEX QTrap 5500 massspectrometer coupled with an Agilent 1290 HPLC Infinity series, a CTCPAL chilled autosampler, all controlled by Analyst software. Afterseparation on a C18 reverse phase HPLC column (Acquity UPLC HSS T3, 1.8,2.1×50 mm) using an acetonitrile-water gradient system, peaks wereanalyzed by mass spectrometry (MS) using ESI ionization in Q1 scan mode.LC conditions are shown in Table 3 below.

TABLE 3 LC Gradient Time Flow rate % A % B (min) (mL/min) Mobile PhaseMobile Phase 0.05 0.6 100 2 5.0 0.6 60 40 6.0 0.6 5 95 6.4 0.6 5 95 6.410.6 100 0 6.8 0.6 100 0

Solution A: H₂O with 0.1% formic acid; Solution B: Acetonitrile with0.1% formic acid

Table 4 shows experimental parameters for metabolite profiling.

TABLE 4 Metabolite Profiling in Hepatocytes: Experimental ConditionsTest Test Hepatocyte Cell Time Points Analytical Article conc. sourcecount Profiled method NS2 (all 4 species) 3 μM Rat, dog, 1 × 10⁶ viable0 and 120 min LC-MS/MS D-NS2 (human only) monkey (cyno) cells/mL andhuman

Experimental Procedure

The test article was incubated in duplicate with primary, cryopreservedhepatocytes at 37° C. The cells were thawed, viable cells counted, andequilibrated according to the supplier's directions. After 30 minequilibration at 37° C. with gentle agitation, the test compound wasadded into the cells to give the desired final concentration of 3 μM.The cell suspension was incubated at 37° C. as described above. At theindicated times, samples were removed and mixed with an equal volume ofice-cold stop solution (methanol).

In parallel, a blank hepatocyte sample in the absence of test agent wasincubated for 120 min and was used as a control to show the presence ofpeaks derived from the hepatocytes. Stopped reactions were incubated atleast ten minutes on ice, and an additional volume of water was added.The samples are centrifuged to remove precipitated protein, and thesupernatants were analyzed by LC-MS/MS.

A full scan mass spectrum (100-800 m/z) in both positive and negativeionization modes were run across the gradient to look for the presenceof potential metabolites (novel masses and known Phase I and IImetabolites such as: oxidation, sulfation, di-oxidation,dehydrogenation, sulfation+oxidation, glucuronidation,oxidation+glucuronidation, and glutathione conjugation). The massspectrometry method is shown below in Table 5.

TABLE 5 Mass Spectrometry Method Development Test ESI Full Scan ArticleMW Polarization Mass Range NS2 236.7 Positive 100-800 CoteRX micronizedLot 093 (Origin Lot BR-NS2-11-01) D-NS2 242.7 Positive 100-800 (Lot1509342002) D-NS2: deuterated N32 (C12H7ClN2Od6)

Results

It was surprisingly found that deuteration of NS2 greatly reduced thenumber and amounts of metabolites in human hepatocytes. As summarized inTables 6A and 6B below, the proteo- (non-deuterium enriched) form of NS2was significantly metabolized over the course of only 120 min by rat,dog, monkey, or human hepatocytes. For example, dog hepatocytesmetabolized NS2 into two different mono-oxidation products (M1 and M2)and two different glutathione (GSH) conjugates (M5 and M6), as evaluatedby LC retention times and mass spectrometry data (see FIG. 3). Cynomonkey hepatocytes metabolized NS2 into a mono-oxidation metabolite(M1), four oxidation+glucuronidation metabolites (M3, M4, M7, and M9),and a glucuronidation metabolite (M8) (see FIG. 2). Human hepatocytesmetabolized NS2 into a mono-oxidation metabolite (M1), twooxidation+glucuronidation metabolites (M7 and M9), and a glucuronidationmetabolite (M8) (see FIG. 1).

TABLE 6A Metabolite Profiling of NS2 in Hepatocytes: Data SummaryRetention Observed Metabolites (T-120 min) Time m/z Possible CynoAnalyte (min) m/z shift Biotransformation Rat Dog Monkey Human NS2 4.4237 — Parent NA NA NA NA M1 2.1 253  +16 mono-oxidation ++++ ++++ ++++++++ M2 2.9 253  +16 mono-oxidation + M3 3.0 429 +192 oxidation + +glucuronidation M4 3.2 429 +192 oxidation + ++ glucuronidation M5 3.5542 +305 GSH-conjugation + M6 3.7 542 +305 GSH-conjugation ++ M7 1.7 429+192 oxidation + trace ++ + glucuronidation M8 3.98 413 +176glucuronidation trace ++ ++ M9 3.9 429 +192 oxidation + + +glucuronidation m/z: Mass-to-Charge ratio of analyte NA = Not applicableRelative degree of observed metabolite formation is denoted by “+”, with++++ being the most abundant metabolite (assuming that the ionizationpotential of the parent is similar to that of the metabolites)

TABLE 6B Peak Areas of Observed Metabolites Peak Areas ObservedMetabolites (T-120 mm) Cyno Analyte Rat Dog Monkey Human NS2 3.14E+084.79E+07 1.74E+08 4.41E+08 M1 4.25E+08 2.83E+08 4.47E+08 2.57E+08 M21.58E+07 M3 7.50E+06 M4 1.92E+07 M5 1.69E+07 M6 7.67E+07 M7 trace1.35E+07 1.62E+07 M8 trace 3.15E+07 4.67E+07 M9 1.22E+07 1.51E+07

Mass spectral analysis of NS2, each metabolite M1-M9, deuterated NS2,and the metabolite produced in human hepatocytes from deuterated NS2 areshown in FIGS. 6-17.

In contrast to non-deuterium-enriched NS2, exposure ofdeuterium-enriched NS2 (i.e., compound I-1, or NS2-D6 in Table 7) tohuman hepatocytes for 120 min resulted in mono-oxidation metabolite M1as the sole detectable metabolite (see Table 7 and FIGS. 5 and 17). Theamount of M1 produced was also greatly reduced relative to that producedwhen non-deuterated NS2 was exposed to human hepatocytes (compare FIGS.1 and 5). Such a dramatic reduction in metabolite production could nothave been predicted in advance.

TABLE 7 Metabolite Profiling of Deuterated NS2 in Human Hepatocytes:Data Summary Retention Observed Metabolites Time m/z Possible (T-120min) Analyte (min) m/z shift Biotransformation Human NS2-D6 4.4 243 —Parent NA M1 2.1 259 +16 mono-oxidation ++ m/z: Mass-to-Charge ratio ofanalyte NA = Not applicableRelative degree of observed metabolite formation is denoted by “+”, with++++ being the most abundant metabolite (assuming that the ionizationpotential of the parent is similar with the metabolites)Note: Based on the number of observed metabolites, deuterated-NS2exhibited less metabolism over the course of 2 hours relative to thenon-deuterated NS2 molecule.

The results shown in Table 7 are also surprising in that the reductionin the number and amount of metabolites may not simply be due todeuterium incorporation making an enzymatically-catalyzedrate-determining step more difficult. Rather, the observed m/z of 259 isconsistent with all six deuterium atoms being retained in themono-oxidation product. Without wishing to be bound by theory, this mayindicate that the deuterium enrichment is influencing metabolism at aremote location on the molecule, and perhaps not simply through, e.g., aprimary kinetic isotype effect.

References: 1) McGinnity, D. F. et al. (2004). “Evaluation of fresh andcryopreserved hepatocytes as in vitro drug metabolism tools for theprediction of metabolic clearance.” Drug Metab. Dispos.32(11):1247-1253. 2) Sahi, J. et al. (2010). “Hepatocytes as a tool indrug metabolism, transport and safety evaluations in drug discovery.”Current Drug Discov. Technol. 7(3): 188-198.

Example 12: Evaluation of Dose Responses for Protective Activity fromHydrogen Peroxide Toxicity in Dissociated Hippocampal Cultures for NS2(i.e. Non-Deuterated NS2)

Experimental Plan for NS2 Dose Response Evaluation for Protection fromHydrogen Peroxide Toxicity:

A. Test Agent: NS2 FW: 236

1. Source 1: CoreRx lot 093-FOR CNS2; amount used: 6.4 mg; micro-milledsample (average particle size is about 16 micron); was derived fromJ-Star Lot BR-NS2-11-01

2. Source 2: J-Star lot BR-NS2-1; amount used: 6 mg; non-milled sample

B. Formulations and Stock Solution Preparation

Two types of formulation were compared: dimethyl sulfoxide (DMSO) andCaptisol®

1. Captisol® formulation: Captisol® was dissolved at 5 mg/ml (i.e., at0.5%) in Dulbecco's Phosphate Buffered Saline (DPBS). A 10 mM NS2 stocksolution (stock A) was prepared by dissolving 1 mg (4.24 mol) into 0.42ml of the Captisol® solution to give an initial stock of 10 Knol/m1 or10 mM (i.e., 2.38 mg/mL). The stock solution was clear after vortexmixing.

2. DMSO formulation: DMSO was used as a comparator to Captisol®. Five mg(21.12 mol) of NS2 was dissolved in 0.21 mL of DMSO for a 100 mM stocksolution (i.e., 23.8 mg/mL). The 100 mM NS2 DMSO formulation was clear.Log dilutions were done with DPBS. Upon dilution 1:10 to 10 mM withDPBS, the solution became cloudy, but, cleared after extensive vortexmixing. In general, our benchmark concentration goal for DMSO in primaryneuronal cultures is less than 0.1%, to avoid pharmacological actions ofDMSO. Note that 1% DMSO was used for the 1 mM test concentration of NS2.No apparent toxicity was observed in the assays in the 5 hr test (seeresults from Experiment 9 below).

C. Details on the Preparation of Stock Solutions

NS2 in Captisol®:

1. Stock A was 10 mM NS2 in 0.5% Captisol®. Added 10 μl into 100 μl fora final concentration of 1 mM NS2, in 0.05% Captisol®, in each well.

2. Stock B was prepared by adding 50 μl of stock A to 450 μl of DPBS toyield a 1 mM solution of NS2. Added 10 μl into 100 μl DPBS for a finalconcentration of 100 μM NS2 in 0.005% Captisol®.

3. Stock C was prepared by adding 50 μl of stock B into 450 μl of DPBSto yield a 100 μM solution of NS2. Added 10 μl into 100 μl DPBS for afinal concentration of 10 μM NS2 in 0.0005% Captisol®.

4. Stock D was prepared by adding 50 μl of stock C into 450 μl of DPB Sto yield a 10 μM solution of NS2. Added 10 μl into 100 μl DPB S for afinal concentration of 1 μM NS2 in 0.00005% Captisol®.

5. Stock E was prepared by adding 50 μl of stock D into 450 μl of DPBSto yield a 1 μM solution of NS2. Added 10 μl into 100 μl DPBS for afinal concentration of 0.1 μM NS2 in 0.000005% Captisol®.

NS2 in DMSO:

1. Stock A was 100 mM NS2 in 100% DMSO.

2. Stock B was prepared by adding 50 μl of stock A into 450 μl of DPBSfor a final concentration of 10 mM NS2. Added 10 μl into 100 μl DPBS toyield a final concentration of 1 mM NS2 in 1% DMSO.

3. Stock C was prepared by adding 50 μl of stock B into 450 μl of DPBSfor a final concentration of 1 mM NS2. Added 10 μl into 100 μl DPB S toyield a final concentration of 100 μM NS2 in 0.1% DMSO.

4. Stock D was prepared by adding 50 μl of stock C into 450 μl of DPBSfor a final concentration of 100 μM NS2. Added 10 μl into 100 μl DPBS toyield a final concentration of 10 μM NS2 in 0.01% DMSO.

5. Stock E was prepared by adding 50 μl of stock D into 450 μl of DPBSfor a final concentration of 10 μM NS2. Added 10 μl into 100 μl DPBS toyield a final concentration of 1 μM NS2 in 0.001% DMSO.

6. Stock F was prepared by adding 50 μl of stock E into 450 μl of DPBSfor a final concentration of 1 μM NS2. Added 10 μl into 100 μl DPBS fora final concentration of 0.1 μM NS2 in 0.0001% DMSO.

In all cases, 10 μl of the appropriate dilution was added to 100 μl fora total volume of 110 μl in the well.

D. Culture Conditions Designed to Detect NS2-Mediated Neuroprotectionfrom Oxidative Stress Associated with Hydrogen Peroxide

1. Rat hippocampal cultures were prepared as previously described(Brenneman D E, Smith G R, Zhang Y, Du Y, Kondaveeti S K, Zdilla M J,Reitz A B. (2012) J. Molecular Neuroscience, 47:368-379). Under theseconditions, the cultures are at least 90% neuronal. The most abundantnon-neuronal cells are astrocytes.

2. All cultures were prepared into a 96-well format at a plating densityof 10K cells per well. Cultures were treated between day 10 and day 21after dissociation of E18 hippocampal tissue. For these experiments, allplates were treated on day 13. In all experiments, the hydrogen peroxidewas added to the cultures 10 minutes after treatment with NS2 orcannabidiol (CBD). For each treatment condition, the number ofreplicates was five.

3. All cultures were plated in B27/Neural Basal Medium. On the day oftreatment, all cultures were given a complete change of medium intoB27/Neural Basal Medium without antioxidants.

4. As previously determined (Brenneman et al., 2012), 10 μM hydrogenperoxide was used to produce toxicity and oxidative stress. As describedpreviously [Jarrett, S G, Liang, L-P, Hellier, J L, Staley, K J andPatel, M. (2008) Neurobiol. Dis 30(1): 130-138], 10 μM hydrogen peroxidehas been observed in the hippocampus of rats with a kainate model ofstatus epilepticus.

5. The positive control used in all studies was 10 μM cannabidiol (CBD),a known antioxidant agent [Hampson et al. (1998), Proc. Nat. Acad. Sci95:8268-8273] that is protective against oxidative stress in primaryneurons [Brenneman, D E, Petkanas, D and Kinney, W. A. (2014) AnnualSymposium on the Cannabinoids, page 129].

6. Neither the negative control wells, the hydrogen peroxide wells, northe positive control wells contained any drug vehicle.

E. Assays

Both assays used in this study have been described in detail [BrennemanD E, Smith G R, Zhang Y, Du Y, Kondaveeti S K, Zdilla M J, Reitz A B.(2012) J. Molecular Neuroscience, 47:368-379].

1. The CFDA neuronal viability assay. In this assay, the CFDA dye istaken up by all live cells and cleaved by esterases to releasefluorescein. The neuronal specificity is achieved because neurons cannotremove this dye, whereas efflux of the dye from non-neuronal cells canoccur over time. After washing away the extracellular dye, the cultureswere read in a fluorimeter; intracellular dye intensity is proportionalto the live neuronal population. Original reference: Petroski, R E andGeller H M. (1994) Selective labeling of embryonic neurons cultures onastrocyte monolayers with 5(6)-carboxyfluorescein diacetate (CFDA) J.Neurosci. Methods 52:23.32. The mean control level for each experimentis shown as a longdashed reference line.

2. A cell death assay, using propidium iodide, was conductedsimultaneously with the CFDA assay in the same well. This dye isexcluded from live cells and binds to the DNA of dead cells. The assaydetects both necrotic and apoptotic cell death; it does not distinguishbetween neuronal cell death and non-neuronal cell death. See Sarafian TA, Kouyoumjian S, Tashkin D, Roth M D. (2002) Tox. Letters. 133:171-179. The mean control level is shown as a medium-dashed referenceline.

3. Reagents used:

-   -   a. Hydrogen Peroxide solution, 30 wt %; Sigma-Aldrich        (216736-100 ml, Lot MKBV382V)    -   b. Captisol® (Lot 17CX01-HQ-00088) provided by Aldeyra        Therapeutics    -   c. Dimethyl Sulfoxide; Sigma-Aldrich (472301-100 ml) Batch 21096        JK    -   d. Propidium Iodide Sigma-Aldrich (P4864-10 ml; 1 mg/ml solution        in water)    -   e. CFDA [5(6)-Carboxyfluorescein Diacetate] Sigma-Aldrich        Product Number: 21879-100 mg-F    -   f. Cannabidiol solution, 10 mg/ml in ethanol; Sigma-Aldrich        Product Number: 90899-1 ml    -   g. Dulbecco's Phosphate Buffered Saline. Gibco (14190-144) Lot        1165767

4. Data Analyses

-   -   a. Data Acquisition: Data were stored on Advanced Neural        Dynamics computers for analyses. Data acquisition was performed        on Cytofluor Fluorimeter and transferred to Excel spreadsheet        for analysis with Sigma Plot 11.    -   b. Statistical Analysis: All data were statistically analyzed by        an Analysis of Variance with the Multiple Comparisons versus        Control Group (Holm-Sidak) method. Statistical significance was        taken at the P<0.05 level. In all cases, comparisons were made        to the negative control (10 μM hydrogen peroxide treatment).    -   c. Methodology for EC₅₀ determination:        -   i. A broad concentration range was chosen to screen NS2 in            an EC₅₀ potency analysis. A log-based concentration series            from 0.1 μM to 1 mM was used, recognizing that further            analysis involving half-log concentration may be necessary            to assess EC₅₀s.        -   ii. A nonlinear regression analysis was used to determine            the equation of the line that best fits the data. (Four            parameter Logistic curve)        -   iii. Based on the Logistic equation below, the EC₅₀s for            neuroprotection were calculated and plotted by SigmaPlot 11            to determine the concentration required to produce            half-maximal responses for both assays. Drop lines were used            to show the axes intersections determining the EC₅₀.            -   Four Parameter Logistic Equation

$\;{y = {\min + \frac{\max - \min}{1 + \left( \frac{x}{{EC}\; 50} \right)^{- {Hillslope}}}}}$

-   -   -   This results in a typical dose-response curve with a            variable slope parameter. It is sometimes abbreviated as            4PL. The four parameters are: min (bottom of the curve), max            (top of the curve). EC₅₀=Concentration of ligand that            produced 50% of maximal effective response.

5. Summary of Results

Table 8 shows a summary of protection studies for NS2 (i.e.,non-deuterated NS2) in rat hippocampal cultures.

TABLE 8 Summary of Protection Studies for N52 in Rat HippocampalCultures Full No Efficacy effect Formu- Concen- Concen- Assay Compoundlation tration** tration EC50 ± SE CFDA NS2 CoreRx Captisol ® 100 μM 1μM 6.8 ± 3.6 μM CFDA NS2 CoreRx DMSO 100 μM 1 μM 9.8 μM * CFDA NS2J-Star Captisol ® 100 μM 1 μM 9.1 ± 2.8 μM CFDA NS2 J-Star DMSO 100 μM 1μM 2.6 μM * PI NS2 CoreRx Captisol ®  10 μM 1 μM 1.3 μM * PI NS2 CoreRxDMSO  10 μM 1 μM 1.3 μM * PI NS2 J-Star Captisol ®  10 μM 1 μM 2.8 μM *PI NS2 J-Star DMSO  10 μM 1 μM 1.1 μM * CFDA None Captisol ® Not activePI None DMSO Not active * Because of the steep nature of the logisticcurve observed for these data sets, further analysis using half-logconcentrations may be necessary to determine the EC₅₀ under theseconditions. The posted values should be considered estimates.**Concentration of test agent showing assay response levels notsignificantly different from that of no treatment controls.

6. Graphical Analyses of Experimental Findings and Raw Data

a. Experiment 1: Dose response to micro-milled NS2 (CoreRx) inCaptisol®. Effect on neuronal viability after co-treatment with 10 μMhydrogen peroxide.

-   -   i. NS2 Source: CoreRx, micro-milled    -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®    -   iii. Assay: CFDA    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   viii. Conclusions: The EC₅₀ was observed at 6.8 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 100 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity at 100μM in this assay. Results are shown in Table 9 and FIGS. 18 and 19.

TABLE 9 Effect on Neuronal Viability After Co-Treatment with 10 μMHydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 +HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10μM 45216 45216 45216 55866 41262 31538 31107 24829 42919 53439 4521647620 27827 24829 29842 27827 47620 50134 35167 45216 48549 30050 2782729430 47620 52763 53439 45216 39139 23428 19585 25008 48706 42919 3540538622 31107 38622 26294 28320 Mean 46416* 48894* 42889* 46508* 37577*29693 26931 27083 Std Error  1044  2078  3448  2778  3697  2703  2014 921 P value * <0.001 <0.001 <0.001 <0.001 <0.002 N.S. N.S. N.S. % ofControl 100 ± 2 105 ± 4 92 ± 07 100 ± 6 80 ± 8 64 ± 6 58 ± 4 58 ± 2 *Significantly different from treatment with 10 μM hydrogen peroxidealone. N.S.: Not significantly different from treatment with 10 μMhydrogen peroxide alone.

b. Experiment 2: Dose response to micro-milled NS2 (CoreRx) inCaptisol®. Effect on cell death after co-treatment with 10 μM hydrogenperoxide.

-   -   i. NS2 Source: CoreRx (micro-milled)    -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®    -   iii. Assay: Propidium Iodide    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   viii. Conclusions: The EC₅₀ was observed at 1.3 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity withthis assay. These data suggest that the protective effect against celldeath may be slightly more potent than that observed for neuronalviability. Results are shown in Table 10 and FIGS. 20 and 21.

TABLE 10 Effect of NS2 (CoreRx) on Cell Death in Hippocampal CulturesAfter Co- treatment with 10 μM Hydrogen Peroxide for 5 hours StatisticalCBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP AnalysisControl 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 140 144 246 144 180319 246 315 180 121 184 180  57 246 246 306 137 192 180 192 246 254 246209 144 117 188 246 180 246 246 346 180 180 217 137 180 267 323 267 Mean156* 151* 203* 180* 169* 266 261 289 Std Error  10  15  13  20  31  14 15  24 P value * <0.001 <0.001 <0.003 <0.001 <0.001 N.S. N.S. N.S. % ofControl 100 ± 6 97 ± 9 130 ± 8 115 ± 13 108 ± 20 171 ± 9 167 ± 10 185 ±15 * Significantly different from treatment with 10 μM hydrogen peroxidealone. N.S.: Not significantly different from treatment with 10 μMhydrogen peroxide alone.

c. Experiment 3: Dose response to micro-milled NS2 (CoreRx) in DMSO.Effect on neuronal viability after co-treatment with 10 μM hydrogenperoxide.

-   -   i. NS2 Source: CoreRx (micro-milled)    -   ii. Formulation: Initial stock was formulated in 100% DMSO    -   iii. Assay: CFDA    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   viii. Conclusions: The EC₅₀ was observed at 9.8 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 100 μM NS2.

The use of DMSO as a formulation produced an EC₅₀ that was very similarto that observed with Captisol®. NS2 is fully neuroprotective againsthydrogen peroxide toxicity in this assay. Results are shown in Table 11and FIGS. 22 and 23.

TABLE 11 Effect of NS2 (CoreRx; in DMSO) on Neuronal Viability After Co-Treatment with 10 mM Hydrogen Peroxide for 5 hours Statistical CBD + HPNS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 47420 49934 38422 42719 38422 2076523428 20844 38939 48038 36415 40792 32661 32886 26094 31447 40792 4271942719 42439 32661 32886 27627 31338 39199 42719 39139 53580 32661 2322825189 24899 48706 38422 36415 37108 34496 23400 29230 23228 Mean 43011*44366* 38622* 43328* 34180* 26633 26314 26351 Std Error  2097  2064 1158  2751  1118  2595  997  2157 P value * <0.001 <0.001 <0.001 <0.001<0.009 N.S. N.S. N.S. % of Control 100 ± 5 103 ± 5 90 ± 3 101 ± 6 79 ± 362 ± 6 61 ± 2 61 + 5 * Significantly different from treatment with 10 μMhydrogen peroxide alone. N.S.: Not significantly different fromtreatment with 10 μM hydrogen peroxide alone.

d. Experiment 4: Dose response to micro-milled NS2 (CoreRx) in DMSO.Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: CoreRx (micro-milled)    -   ii. Formulation: Initial stock was formulated in 100% DMSO    -   iii. Assay: Propidium Iodide    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   viii. Conclusions: The EC₅₀ was observed at 1.3 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity withthis assay. These data suggest that the protective effect against celldeath may be slightly more potent than that observed for neuronalviability. Results are shown in Table 12 and FIGS. 24 and 25.

TABLE 12 Effect of NS2 (CoreRx in DMSO) on Cell Death in HippocampalCultures After Co-Treatment with 10 μM Hydrogen Peroxide StatisticalCBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP AnalysisControl 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 117 180 204 125 180254 246 315 133 180 180 180 188 263 263 246 144 117 221 180 192 254 254346 180 125 188 192 117 254 315 246 129 117 180 117 137 315 259 259 Mean141* 144* 195* 159* 163* 268 267 282 Std Error  11  15  8  16  15  12 12  20 P value * <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. N.S. % ofControl 100 ± 8 102 ± 11 138 ± 6 113 ± 11 115 ± 11 190 ± 9 189 ± 9 200 ±14 * Significantly different from treatment with 10 μM hydrogen peroxidealone. N.S.: Not significantly different from treatment with 10 μMhydrogen peroxide alone.

e. Experiment 5: Dose response to non-milled NS2 (J-Star) in Captisol®.Effect on neuronal viability after co-treatment with 10 μM hydrogenperoxide.

-   -   i. NS2 Source: J-Star (non-milled)    -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®    -   iii. Assay: CFDA    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   viii. Conclusions: The EC₅₀ was observed at 9.1 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 100 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity withthis assay. Results are shown in Table 13 and FIGS. 26 and 27.

TABLE 13 Dose Response Data for Non-Milled NS2 (J-Star) in Captisol ®Showing Effect on Neuronal Viability after Co-Treatment with 10 μMHydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 +HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10μM 40722 36415 38422 36415 37407 22135 21242 25171 41535 38422 4072231538 36861 24829 26094 21889 41198 41884 38622 29230 32861 26094 2205323428 40992 43711 43200 42919 26756 28021 22089 19885 36415 38809 3661547620 25171 20609 29230 21162 Mean 40172 * 40858 * 39516 * 37544 *31811 * 24338 24142 22307 Std Error  949  5136  1128  3442  2525  1335 1528  917 P value * <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. N.S. %of Control 100 ± 2 102 ± 12 98 ± 3 93 ± 9 79± 6 61 ± 3 60 ± 4 56 ± 2 *Significantly different from treatment with 10 μM hydrogen peroxidealone. N.S.: Not significantly different from treatment with 10 μMhydrogen peroxide alone.

f. Experiment 6: Dose response to non-milled NS2 (J-Star) in Captisol®.Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: J-Star (non-milled)    -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®    -   iii. Assay: Propidium Iodide    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   viii. Conclusions: The EC₅₀ was observed at 2.8 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity withthis assay. These data suggest that the protective effect against celldeath may be slightly more potent than that observed for neuronalviability. Results are shown in Table 14 and FIGS. 28 and 29.

TABLE 14 Dose Response Data for Non-Milled NS2 (J-Star) in Captisol ®Showing Effect on Cell Death after Co-Treatment with 10 μM HydrogenPeroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 +HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 140 180180 129 117 184 204 271 144 144 133 129 196 315 180 323 140 117 172 125117 337 246 246 117 121 180 180 180 180 350 246 164 200 180 133 188 192180 263 Mean 141 * 152 * 169 * 139 * 160 * 242 232 270 Std Error  7  16 9  10  18  35  32  14 P value * <0.001 <0.001 <0.001 <0.001 <0.001 N.S.N.S. N.S. % of Control 100 ± 5 108 ± 11 120 ± 6 99 ± 7 113 ± 12 172 ± 24165 ± 22 191 ± 10 *Significantly different from treatment with 10 μMhydrogen peroxide alone. N.S.: Not significantly different fromtreatment with 10 μM hydrogen peroxide alone.

g. Experiment 7: Dose response to non-milled NS2 (J-Star) in DMSO.Effect on neuronal viability after co-treatment with 10 μM hydrogenperoxide.

-   -   i. NS2 Source: J-Star (non-milled)    -   ii. Formulation: Initial stock was formulated in 100% DMSO    -   iii. Assay: CFDA    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   viii. Conclusions: The EC₅₀ was observed at 2.6 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 100 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity withthis assay. Results are shown in Table 15 and FIGS. 30 and 31.

TABLE 15 Dose Response Data for Non-Milled NS2 (J-Star) in DMSO ShowingEffect on Neuronal Viability after Co-Treatment with 10 μM HydrogenPeroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 +HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 4072247620 45216 42919 31214 20809 23600 19585 42919 34696 39269 38622 3376829430 25371 24829 42919 48238 26576 38622 33425 23257 31107 25462 4334241535 26294 26294 32194 34696 22089 22089 38622 36615 45807 52763 4610529430 21926 23772 Mean 41705* 41741* 36632* 39844* 35341* 27524 2481923147 Std Error 897 2763 4318 4260 2729 2470 1690 1058 P value * <0.001<0.001 <0.002 <0.001 <0.004 N.S. N.S. N.S. % of Control 100 ± 2 100 ± 788 ± 10 96 ± 10 85 ± 7 66 ± 6 60 ± 4 56 + 3 *Significantly differentfrom treatment with 10 μM hydrogen peroxide alone. N.S.: Notsignificantly different from treatment with 10 μM hydrogen peroxidealone.

h. Experiment 8: Dose response to non-milled NS2 (J-Star) in DMSO.Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: J-Star (non-milled)    -   ii. Formulation: Initial stock was formulated in 100% DMSO    -   iii. Assay: Propidium Iodide    -   iv. Toxin: 10 μM hydrogen peroxide    -   v. Duration of treatment: 5 hours    -   vi. Growth medium: B27/neurobasal medium without antioxidants    -   vii. Culture matrix: poly-L-lysine    -   vii. Conclusions: The EC₅₀ was observed at 1.1 μM; full efficacy        relative to controls (CBD+HP and no-treatment control) was        observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity withthis assay. These data suggest that the protective effect against celldeath may be slightly more potent than that observed for neuronalviability. Results are shown in Table 16 and FIGS. 32 and 33.

TABLE 16 Dose Response Data for Non-Milled NS2 (J-Star) in DMSO ShowingEffect on Cell Death after Co-Treatment with 10 μM Hydrogen PeroxideStatistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HPAnalysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 180 180 180125 180 246 246 254 117 133 204 125 180 196 315 246 180 117 133 246 172246 204 319 164 129 117 246 180 188 350 246 144 184 250 133 192 250 259259 Mean 157* 161* 177* 175* 181* 225 275 265 Std Error 12 12 24 29 3 1426 14 P value * <0.001 <0.001 <0.002 <0.002 <0.003 N.S. N.S. N.S. % ofControl 100 ± 8 103 ± 8 113 ± 15 111 ± 18 115 ± 2 143 ± 9 175 ± 17 169 ±9 *Significantly different from treatment with 10 μM hydrogen peroxidealone. N.S.: Not significantly different from treatment with 10 μMhydrogen peroxide alone.

i. Experiment 9: Dose response to Formulation vehicles. Effect onneuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   i. Test agents: Captisol® [Cap; (5 mg/ml i.e., 0.5%)] and 1%        DMSO (abbreviated to DM; 1% was the highest concentration used).        The amounts used for the formulations were matched to those used        in the NS2 studies.    -   ii. Assay: CFDA    -   iii. Toxin: 10 μM hydrogen peroxide    -   iv. Duration of treatment: 5 hours    -   v. Growth medium: B27/neurobasal medium without antioxidants    -   vi. Culture matrix: poly-L-lysine    -   vii. Conclusions: There was no detectible effect on neuronal        viability from either Captisol® or DMSO when tested under the        same conditions as for NS2. Results are shown in Tables 17 and        18 and FIG. 34.

TABLE 17 Dose Response Data for Formulation Vehicles Showing Effect onNeuronal Viability after Co-Treatment with 10 μM Hydrogen PeroxideStatistical CBD + HP Cap + HP Cap + HP Cap + HP Cap + HP Cap + HP HPAnalysis Control 10 μM 500 μγ/ml 50 μg/ml 5 μg/ml 500 pg/ml 50 pg/ml 10μM 52563 52563 24629 26471 23833 23228 27627 24629 34496 34496 2923024629 25171 28219 23833 26094 49934 42719 20687 27627 23833 25171 2471823228 38422 42719 31107 26188 27627 26471 26094 21889 45016 45216 2974623228 29230 23572 26094 27627 Mean 44086* 43543* 27080 25629 25939 2533225673 24693 Std Error 3398 2893 1934 767 1076 927 651 1014 P value *<0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 8 99 ± 761 ± 4 58 ± 2 59 ± 2 57 ± 2 58 ± 2 56 ± 2 *Significantly different fromtreatment with 10 μM hydrogen peroxide alone. N.S.: Not significantlydifferent from treatment with 10 μM hydrogen peroxide alone.

TABLE 18 Dose Response Data for Formulation Vehicles Showing Effect onNeuronal Viability after Co-Treatment with 10 μM Hydrogen PeroxideStatistical CBD + HP DM + HP DM + HP DM + HP DM + HP DM + HP HP AnalysisControl 10 μM 1% 0.1% .01% .001% .0001% 10 μM 52563 52563 24629 3166524629 21003 23228 22218 34496 34496 24629 25171 26094 32661 28219 2342849934 42719 24629 32886 24629 29230 19685 24899 38422 42719 27627 2208926094 18215 19385 21889 45016 45216 27627 29333 24629 18215 21971 26482Mean 44086* 43543* 25828 28229 25215 23865 22498 23783 Std Error 33982893 734 2022 359 2985 1599 857 P value * <0.001 <0.001 N.S. N.S. N.S.N.S. N.S. N.S. % of Control 100 ± 8 99 ± 7 59 ± 2 64 ± 5 57 ± 1 54 ± 751 ± 4 54 ± 2 *Significantly different from treatment with 10 μMhydrogen peroxide alone. N.S.: Not significantly different fromtreatment with 10 μM hydrogen peroxide alone.

j. Experiment 10: Dose response to Formulation vehicles. Effect on celldeath after co-treatment with 10 μM hydrogen peroxide.

-   -   i. Test agents: Captisol® [CP (5 mg/ml i.e., 0.5%)] and DMSO        (abbreviated to DM; 1% was the highest used)    -   ii. Assay: Propidium Iodide    -   iii. Toxin: 10 μM hydrogen peroxide    -   iv. Duration of treatment: 5 hours    -   v. Growth medium: B27/neurobasal medium without antioxidants    -   vi. Culture matrix: poly-L-lysine    -   vii. Conclusions: There was no detectible effect on cell death        from either Captisol® or DMSO when tested under the same        conditions as for NS2. Results are shown in Tables 19 and 20 and        FIG. 35.

TABLE 19 Dose Response Data for Formulation Vehicles Showing Effect onCell Death after Co-Treatment with 10 μM Hydrogen Peroxide StatisticalCBD + HP Cap + HP Cap + HP Cap + HP Cap + HP Cap + HP HP AnalysisControl 10 μM 500 μg/ml 50 μg/ml 5 mg/ml 500 pg/ml 50 pg/ml 10 μM 125140 246 246 246 246 229 263 144 133 246 254 250 246 229 188 117 180 263180 140 180 246 246 180 184 250 350 246 315 246 246 144 156 250 315 288319 246 250 Mean 142* 159* 251 269 234 261 239 239 Std Error 11 10 3 2925 26 4 13 P value * <0.001 <0.003 N.S. N.S. N.S. N.S. N.S. N.S. % ofControl 100 ± 8 112 ± 7 177 ± 2 189 ± 20 165 ± 18 184 ± 18 168 ± 3 168 ±9 Cap = Captisol ® vehicle. *Significantly different from treatment with10 μM hydrogen peroxide alone. N.S.: Not significantly different fromtreatment with 10 μM hydrogen peroxide alone.

TABLE 20 Dose Response Data for Formulation Vehicles Showing Effect onCell Death after Co-Treatment with 10 μM Hydrogen Peroxide StatisticalCBD + HP DM + HP DM + HP DM + HP DM + HP DM + HP HP Analysis Control 10μM 1% 0.1% .01% .001% .0001% 10 μM 125 140 250 297 276 267 246 267 144133 246 297 267 259 246 254 117 180 315 276 246 246 184 246 180 184 230192 250 246 267 246 144 156 267 276 246 246 225 246 Mean 142* 159* 262268 257 253 234 252 Std Error 11 10 15 19 6 4 14 4 P value * <0.001<0.003 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 8 112 ± 7 185 ±11 189 ± 13 181 ± 4 178 ± 3 165 ± 10 177 ± 3 DM = DMSO vehicle.*Significantly different from treatment with 10 μM hydrogen peroxidealone. N.S.: Not significantly different from treatment with 10 μMhydrogen peroxide alone.

7. Summary of Observations and Conclusions

A. NS2 exhibited neuroprotective activity against hydrogen peroxidetoxicity in the CFDA assay, with both formulations (DMSO and Captisol®)and with both compound batches (CoreRx and J-Star, milled andnon-milled, respectively).

B. The neuroprotective effect of NS2 in the CFDA assay was equal to thecontrol (no HP treatment) and positive control (CBD), indicating fullprotection.

C. Full protection relative to controls was observed with both NS2formulations and NS2 batches at 100 μM NS2 in the CFDA neuronalviability assay, whereas the no effect concentration for bothformulations and batches was 1 μM.

D. Non-linear curve fitting Logistic analyses indicated that NS2 EC₅₀sin the CFDA neuronal viability assay ranged from 3 to 10 μM. The bestavailable EC₅₀ estimate is from the CoreRx NS2 (milled) formulated inCaptisol®, which showed an EC₅₀ of 7±4 μM. All the EC₅₀s from thedifferent NS2 formulations for the CFDA assays fall within this range.It is our conclusion that the two compound batches and the twoformulations are characterized by their substantial similarity.

E. NS2 exhibited protective activity against hydrogen peroxide toxicityin the Propidium Iodide (PI) assay. This was observed with bothformulations and with both compound batches.

F. The protective effect of NS2 in the PI assay was equal to that of thecontrol (no HP treatment) and positive control (CBD), indicating fullprotection.

G. Full protection relative to no-treatment controls was observed withboth NS2 formulations and NS2 batches at 10 μM NS2 in the PI assay,whereas the no effect concentration for both formulations and batcheswas 1 μM. It was a consistent finding that the NS2 response in the celldeath assay exhibited greater potency for full protection than in theCFDA assay. It should be recognized that the cell death assay is notspecific to neurons and may involve non-neuronal cells that are presentin this model CNS system.

H. Non-linear Logistic curve fitting indicated that EC₅₀s for the PIassay ranged from 1.1 to 2.8 μM. However, the steep nature of theLogistic curve with this assay made estimates difficult without themeasurement of half-log concentration responses to help define theinflection point of the curves. The best available estimate is the meanvalue for all PI data, which is 2±1 μM. It is our conclusion that thetwo compound batches and the two formulations were characterized bytheir substantial similarity. Because of the narrow response ranges inthe PI assay, further analysis may be required to refine the EC₅₀estimate. These data suggest that NS2 may be more potent in preventingcell death than in increasing neuronal viability against hydrogenperoxide toxicity.

I. The toxic signal produced by 10 μM hydrogen peroxide was typical of awide variety of oxidative stressors (ethanol, heavy metals, ammoniumacetate, and glutamate) that have been tested in the past, withdecreases from control ranging from 30 to 50%.

J. The positive control (10 μM cannabidiol) was active on every testplate, indicating that the model system was responding in a typicalmanner.

Example 13: Dose Responses for Three Deuterated Compounds AssessingProtective Activity from Hydrogen Peroxide Toxicity in DissociatedHippocampal Cultures

A. Experimental Plan for dose response evaluation for protection fromhydrogen peroxide toxicity.

1. Test Agents:

-   -   a. ALD-6-batch 1 (Legacy ID: NS2-D6 or compound I-1); amount        used: 5.0 mg; MW=242.734    -   b. ALD-5-batch 1 (Legacy ID: D3); amount used: 6.1 mg;        MW=203.24; structure:

-   -   c. ALD-2-batch 1 Legacy ID: D2); amount used: 5.6 mg; MW=203.24;        structure:

2. Formulation and stock solution preparation

-   -   a. Dimethyl sulfoxide (DMSO) at 100% was used for all samples.    -   b. Observations:        -   i. ALD-6: (5 mg, 20.6 μmol) of ALD-6 was dissolved in 0.206            ml of DMSO for a 100 mM stock solution. The 100 mM            ALD-6/DMSO solution was clear. Log dilutions were done with            DPBS. Upon dilution from 1:10 to 10 mM with DPBS, the            solution became cloudy, but cleared after vortex mixing. In            general, the benchmark concentration goal for DMSO in            primary neuronal cultures is less than 0.1%, to avoid            pharmacological effects from the DMSO. Note that 0.3% DMSO            was used for the 300 μM test concentration of all samples.            No apparent toxicity was observed in the assays after the 5            hr test.        -   ii. ALD-5: The 100 mM stock solution of ALD-5 was prepared            by dissolving 6.1 mg (30 μmol) into 0.3 ml of DMSO. The            ALD-5/DMSO mixture was a clear yellow solution. Log            dilutions were done with DPBS. Upon dilution from 1:10 to 10            mM with DPBS, the solution remained a clear yellow solution.        -   iii. ALD-2: The 100 mM stock solution of ALD-2 was prepared            by dissolving 5.6 mg (27.55 μmol) into 0.275 ml of DMSO. The            ALD-2/DMSO mixture was a clear amber solution. Log dilutions            were done with DPBS. Upon dilution from 1:10 to 10 mM with            DPBS, the solution remained a clear amber solution.

c. Details on the preparation of stock solutions: Compound dilution inDMSO/DPBS

-   -   i. Stock A was 100 mM of compound in 100% DMSO.    -   ii. Stock B was prepared by adding 50 μl of stock A into 450 μl        of DPBS for a final concentration of 10 mM. Added 3.3 μl into        100 μl DPBS to yield a final concentration of 300 μM in 0.3%        DMSO.    -   iii. Stock C was prepared by adding 50 μl of stock B into 450 μl        of DPBS for a final concentration of 1 mM. Added 10 μl into 100        μl DPBS to yield a final concentration of 100 μM in 0.1% DMSO.    -   iv. Stock D was prepared by adding 50 μl of stock C into 450 μl        of DPBS for a final concentration of 100 μM. Added 10 μl into        100 μl DPBS to yield a final concentration of 10 μM in 0.01%        DMSO.    -   v. Stock E was prepared by adding 50 μl of stock D into 450 μl        of DPBS for a final concentration of 10 μM. Added 10 μl into 100        μl DPBS to yield a final concentration of 1 μM in 0.001% DMSO.    -   vi. Stock F was prepared by adding 50 μl of stock E into 450 μl        of DPBS for a final concentration of 1 μM. Added 10 μl into 100        μl DPBS for a final concentration of 0.1 μM in 0.0001% DMSO.    -   vii. 10 μl of the appropriate dilution was added to 90 μl for a        total volume of 100 μl in the well.

3. Culture conditions designed to detect neuroprotection from oxidativestress associated with hydrogen peroxide:

-   -   a. Rat hippocampal cultures were prepared as previously        described (Brenneman D E, Smith G R, Zhang Y, Du Y, Kondaveeti S        K, Zdilla M J, Reitz A B. (2012) J. Molecular Neuroscience,        47:368-379). Under these conditions, the cultures are at least        90% neuronal. The most abundant non-neuronal cells are        astrocytes. Rat E18 hippocampal tissue was purchased from Brain        Bits, LLC (Springfield Ill.). Tissue was stored in Hibernate E        medium for transport.    -   b. All cultures were prepared into a 96-well format at a plating        density of 10K cells per well. Cultures were treated between day        10 and day 21 after dissociation of E18 hippocampal tissue. For        these experiments, all plates were treated on day 13. In all        experiments, the hydrogen peroxide was added to the cultures 10        minutes after treatment with the test agent or positive control        (cannabidiol). For each treatment condition, the number of        replicates was five.    -   c. All cultures were plated in B27/Neural Basal Medium. On the        day of treatment, all cultures were given a complete change of        medium into serum-free B27/Neural Basal Medium without        antioxidants.    -   d. As previously determined (Brenneman et al., 2012), 10 μM        hydrogen peroxide was used to produce toxicity and oxidative        stress. As described previously (Jarrett, S G, Liang, L-P,        Hellier, J L, Staley, K J and Patel, M. (2008) Neurobiol. Dis        30(1): 130-138) 10 μM hydrogen peroxide has been observed in the        hippocampus of rats with a kainate model of status epilepticus.    -   e. The positive control used in all studies was 10 μM        cannabidiol, a known antioxidant agent (Hampson et al. (1998),        Proc. Nat. Acad. Sci. 95:8268-8273) that is protective against        oxidative stress in primary neurons (Brenneman, D E, Petkanas, D        and Kinney, W. A. (2014) Annual Symposium on the Cannabinoids,        page 129).    -   f. Neither the negative control wells, the hydrogen peroxide        wells, nor the positive control wells contained any drug        vehicle.

4. Assays:

Both assays used in this study have been described in detail (BrennemanD E, Smith G R, Zhang Y, Du Y, Kondaveeti S K, Zdilla M J, Reitz A B.(2012) J. Molecular Neuroscience, 47:368-379).

-   -   a. The CFDA neuronal viability assay: In this assay, the CFDA        dye is taken up by all live cells and cleaved by esterases to        release fluorescein. The neuronal specificity is achieved        because neurons cannot remove this dye, whereas efflux of the        dye from non-neuronal cells can occur over time. After washing        away the extracellular dye, the cultures were read in a        fluorimeter; intracellular dye intensity is proportional to the        live neuronal population. Original reference: Petroski, R E and        Geller H M, (1994) “Selective labeling of embryonic neurons        cultures on astrocyte monolayers with 5(6)-carboxyfluorescein        diacetate (CFDA),” J. Neurosci. Methods 52:23.32. The mean        control level for each experiment is shown as a long-dashed        reference line.    -   b. A cell death assay, using propidium iodide, was conducted        simultaneously with the CFDA assay in the same well. This dye is        excluded from live cells and binds to the DNA of dead cells. The        assay detects both necrotic and apoptotic cell death; it does        not distinguish between neuronal cell death and non-neuronal        cell death. See Sarafian T A, Kouyoumjian S, Tashkin D, Roth        M D. (2002) Tox. Letters. 133: 171-179. The mean control level        is shown as a red medium-dashed reference line.    -   c. Reagents used        -   i. Hydrogen Peroxide solution, 30 wt %; Sigma-Aldrich            (216736-100 ml, Lot MKBV382V)        -   ii. Dimethyl Sulfoxide; Sigma-Aldrich (472301-100 ml) Batch            21096 JK        -   iii. Propidium Iodide; Sigma-Aldrich (P4864-10 ml; 1 mg/ml            solution in water)        -   iv. CFDA [5(6)-Carboxyfluorescein Diacetate] Sigma-Aldrich            Product Number: 21879-100 mg-F        -   v. Cannabidiol solution, 10 mg/ml in ethanol; Sigma-Aldrich            Product Number: 90899-1 ml        -   vi. Dulbecco's Phosphate Buffered Saline (DPBS). Gibco            (14190-144) Lot 1165767

5. Data Analyses:

-   -   a. Data Acquisition: Data were stored on Advanced Neural        Dynamics computers for analyses. Data acquisition was performed        on Cytofluor Fluorimeter and transferred to Excel spreadsheet        for analysis with Sigma Plot 11.    -   b. Statistical Analysis: All data were statistically analyzed by        an Analysis of Variance with the Multiple Comparisons versus        Control Group (Holm-Sidak) method. Statistical significance was        taken at the P<0.05 level. In all cases, comparisons were made        to the negative control (10 μM hydrogen peroxide treatment).    -   c. Methodology for EC₅₀ determination:        -   i. A broad concentration range was chosen to screen the            compounds in an EC₅₀ potency analysis. A log-based            concentration series from 0.1 μM to 300 μM was used.        -   ii. A nonlinear regression analysis was used to determine            the equation of the line that best fits the data. (Four            parameter Logistic curve)        -   iii. Based on the Logistic equation used in the preceding            Example, the EC₅₀s for neuroprotection were calculated and            plotted by SigmaPlot 11 to determine the concentration            required to produce half-maximal responses for both assays.            Drop lines were used to show the axes intersections            determining the EC₅₀.

B. Summary of Protection Studies for Aldeyra Compounds in RatHippocampal Cultures

TABLE 21 Summary of Protection Studies Data No effect Com- Formu- FullEfficacy Concen- Assay pound lation Concentration* tration EC50 ± SECFDA ALD-6 DMSO 100 μM   1 μM  6.8 ± 1.2 μM CFDA ALD-5 DMSO InactiveInactive Inactive CFDA ALD-2 DMSO Inactive Inactive Inactive PI ALD-6DMSO  10 μM 0.1 μM 0.32 ± 0.03 μM PI ALD-5 DMSO Inactive InactiveInactive PI ALD-2 DMSO Inactive Inactive Inactive *Concentration of testagent showing assay response levels not significantly different fromthat of no treatment controls.

C. Graphical Analyses of Experimental Findings and Raw Data

1. Experiment 1: Dose response of ALD-6. Effect on neuronal viabilityafter co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO    -   b. Assay: CFDA    -   c. Toxin: 10 μM hydrogen peroxide    -   d. Duration of treatment: 5 hours    -   e. Growth medium: B27/neurobasal medium without antioxidants    -   f. Culture matrix: poly-L-lysine    -   g. Conclusions: The EC₅₀ was observed at 6.8±1.2 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 100 μM ALD-6. ALD-6 is fully neuroprotective        against hydrogen peroxide toxicity at 100 μM in this assay.        Results are shown in Table 22 and FIGS. 36 and 37.

TABLE 22 Dose Response Data for ALD-6 Showing Effect on NeuronalViability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD6 +ALD6 + ALD6 + ALD6 + ALD6 + Statistical HP HP HP HP HP HP HP AnalysisControl 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 59551 67121 6577861821 47737 43459 41244 31224 55935 52880 53184 50555 53184 40839 3673232978 61821 55631 53184 57177 45333 33090 31224 43036 59247 64665 5318452880 53184 43340 31224 34419 54201 67958 59551 63181 40839 29547 3154730376 Mean 58151* 57221* 56976* 57123* 48055* 38055 34394 34407 StdError 1363 1977 2522 2449 2368 2845 2007 2268 P value * <0.001 <0.001<0.001 <0.001 <0.001 N.S. N.S. N.S. % of Control 100 ± 2 98 ± 3 98 ± 498 ± 4 83 ± 4 65 ± 5 59 ± 3 59 ± 4 *Significantly different fromtreatment with 10 μM hydrogen peroxide alone. N.S.: Not significantlydifferent from treatment with 10 μM hydrogen peroxide alone.

2. Experiment 2: Dose response effect of ALD-6 on cell death afterco-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO    -   b. Assay: Propidium Iodide    -   c. Toxin: 10 μM hydrogen peroxide    -   d. Duration of treatment: 5 hours    -   e. Growth medium: B27/neurobasal medium without antioxidants    -   f. Culture matrix: poly-L-lysine    -   g. Conclusions: The EC₅₀ was observed at 0.32±0.03 μM; full        efficacy relative to controls (CBD+HP and no-treatment control)        was observed at 10 μM ALD-6. ALD-6 is fully neuroprotective        against hydrogen peroxide toxicity in this assay. These data        suggest that the protective effect against cell death is more        potent than that observed for neuronal viability. Results are        shown in Table 23 and FIGS. 38 and 39.

TABLE 23 Dose Response Data for ALD-6 Showing Effect on NeuronalViability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD6 +ALD6 + ALD6 + ALD6 + ALD6 + Statistical HP HP HP HP HP HP HP AnalysisControl 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 159 119 159 167 179214 225 225 159 159 189 193 183 183 250 366 171 171 189 206 189 223 294294 163 123 147 189 189 183 242 255 171 197 197 135 139 189 356 329 Mean165* 154* 176* 178* 176* 198* 273 294 Std Error 3 15 10 12 9 8 24 25 Pvalue * <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. % of Control100 ± 2 93 ± 9 107 ± 6 108 ± 7 107 ± 5 120 ± 5 165 ± 15 178 ± 15*Significantly different from treatment with 10 μM hydrogen peroxidealone. N.S.: Not significantly different from treatment with 10 μMhydrogen peroxide alone.

3. Experiment 3: Dose response to ALD-5 in DMSO. Effect on neuronalviability after co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO    -   b. Assay: CFDA    -   c. Toxin: 10 μM hydrogen peroxide    -   d. Duration of treatment: 5 hours    -   e. Growth medium: B27/neurobasal medium without antioxidants    -   f. Culture matrix: poly-L-lysine    -   g. Conclusions: There was no statistically significant        neuroprotective activity from ALD-5 against hydrogen peroxide        toxicity from 0.1 to 300 μM. Results are shown in Table 24 and        FIG. 40.

TABLE 24 Dose Response Data for ALD-5 Showing Effect on NeuronalViability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD5 +ALD5 + ALD5 + ALD5 + ALD5 + Statistical HP HP HP HP HP HP HP AnalysisControl 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 55631 58812 4083941143 39126 42756 39043 34930 58508 51551 43036 43743 37102 35588 3873941244 58508 58812 41143 32978 41379 34189 40839 41925 57357 55983 4402836732 34115 38997 38739 33203 54068 62790 42201 33429 32978 35707 3735035588 Mean 56814* 57590* 42249 37605 36940 37447 38942 37378 Std Error865 1858 592 2119 1552 1544 558 1764 P value * <0.001 <0.001 N.S. N.S.N.S. N.S. N.S. N.S. % of Control 100 ± 2 101 ± 3 74 ± 1 66 ± 4 65 ± 3 66± 3 69 ± 1 66 ± 3 *Significantly different from treatment with 10 μMhydrogen peroxide alone. N.S.: Not significantly different fromtreatment with 10 μM hydrogen peroxide alone.

4. Experiment 4: Dose response to ALD-5. Effect on cell death afterco-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO    -   b. Assay: Propidium Iodide    -   c. Toxin: 10 μM hydrogen peroxide    -   d. Duration of treatment: 5 hours    -   e. Growth medium: B27/neurobasal medium without antioxidants    -   f. Culture matrix: poly-L-lysine    -   g. Conclusions: There was no statistically significant        protection from cell death from 0.1 to 300 μM ALD-5. Results are        shown in Table 25 and FIG. 41.

TABLE 25 Dose Response Data for ALD-5 Showing Effect on Cell Death afterCo-Treatment with 10 μM Hydrogen Peroxide CBD + ALD5 + ALD5 + ALD5 +ALD5 + ALD5 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 159 147 294 294 225 294 294 258123 164 293 255 294 294 294 294 159 164 352 250 294 294 366 255 179 83294 250 294 285 225 330 127 123 294 294 320 294 325 294 Mean 149* 136*305 269 285 292 301 286 Std Error 11 15 12 10 16 2 23 14 P value *<0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of 100 ± 7 91 ± 10 205 ± 8181 ± 7 191 ± 11 196 ± 1 202 ± 15 192 ± 9 Control *Significantlydifferent from treatment with 10 μM hydrogen peroxide alone. N.S.: Notsignificantly different from treatment with 10 μM hydrogen peroxidealone.

5. Experiment 5: Dose response to ALD-2. Effect on neuronal viabilityafter co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO    -   b. Assay: CFDA    -   c. Toxin: 10 μM hydrogen peroxide    -   d. Duration of treatment: 5 hours    -   e. Growth medium: B27/neurobasal medium without antioxidants    -   f. Culture matrix: poly-L-lysine    -   g. Conclusions: ALD-2 had no statistically significant        neuroprotection from decreases in neuronal viability in hydrogen        peroxide-treated cultures. Results are shown in Table 26 and        FIG. 42.

TABLE 26 Dose Response Data for ALD-2 Showing Effect on NeuronalViability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD2 +ALD2 + ALD2 + ALD2 + ALD2 + Statistical HP HP HP HP HP HP HP AnalysisControl 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 58877 55631 4303645628 43601 43743 40839 34813 58508 54240 45924 35522 38997 36732 3388540839 56160 50251 45628 36732 50251 43036 40839 43036 55631 62291 3673243176 36732 35522 40839 31224 55807 52880 35048 32311 32978 31331 3481341244 Mean 56997* 55059* 41274 38674 40512 38073 38243 38231 Std Error700 2015 2270 2478 2981 2351 1596 2233 P value * <0.001 <0.001 N.S. N.S.N.S. N.S. N.S. N.S. % of 100 ± 1 97 ± 4 72 ± 4 68 ± 4 71 ± 5 67 ± 4 67 ±3 67 ± 4 Control *Significantly different from treatment with 10 μMhydrogen peroxide alone. N.S.: Not significantly different fromtreatment with 10 μM hydrogen peroxide alone.

6. Experiment 6: Dose response to ALD-2. Effect on cell death afterco-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO    -   b. Assay: Propidium Iodide    -   c. Toxin: 10 μM hydrogen peroxide    -   d. Duration of treatment: 5 hours    -   e. Growth medium: B27/neurobasal medium without antioxidants    -   f. Culture matrix: poly-L-lysine    -   g. Conclusions: There was no statistically significant        protection from cell death produced by hydrogen peroxide after        treatment with ALD-2. Results are shown in Table 27 and FIG. 43.

TABLE 27 Dose Response Data for ALD-2 Showing Effect on Cell Death afterCo-Treatment with 10 μM Hydrogen Peroxide CBD + ALD2 + ALD2 + ALD2 +ALD2 + ALD2 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 143 104 294 294 225 242 266 398159 175 242 225 225 298 294 255 159 139 302 225 293 229 258 242 159 159294 258 329 294 294 294 175 147 320 242 258 258 259 259 Mean 159* 145*290 249 266 264 274 290 Std Error 5 12 13 13 20 14 8 28 P value * <0.001<0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 3 91 ± 8 182 ± 8157 ± 8 167 ± 13 166 ± 9 172 ± 5 182 ± 18 *Significantly different fromtreatment with 10 μM hydrogen peroxide alone. N.S.: Not significantlydifferent from treatment with 10 μM hydrogen peroxide alone.

7. Summary of Observations and Conclusions

-   -   a. ALD-6 exhibited neuroprotective activity against hydrogen        peroxide toxicity in the CFDA assay.    -   b. The neuroprotective effect of ALD-6 in the CFDA assay was not        statistically different from the control (no HP treatment) and        positive control (CBD) values, indicating full protection.    -   c. Full protection relative to controls was observed at 100 μM        ALD-6 in the CFDA neuronal viability assay. The no-effect        concentration for ALD-6 in the CFDA assay was 1 μM.    -   d. Non-linear curve fitting Logistic analyses indicated that the        EC₅₀ of ALD-6 in the CFDA neuronal viability assay was 6.8±1.2        μM.    -   e. ALD-6 exhibited protective activity from cell death from        hydrogen peroxide treatment in the Propidium Iodide (PI) assay.    -   f. The protective effect of ALD-6 from cell death in the PI        assay was not statistically different from that of the control        (no HP treatment) and positive control (CBD) values, indicating        full protection.    -   g. Full protection relative to no-treatment control values was        observed with ALD-6 at 10 μM in the PI assay, whereas the        no-effect concentration was 0.1 μM. ALD-6 response in the cell        death assay exhibited greater potency than in the CFDA assay. It        should be recognized that the cell death assay is not specific        to neurons and may involve non-neuronal cells that are present        in this model CNS system.    -   h. Non-linear Logistic curve fitting indicated that the EC₅₀ for        ALD-6 in the PI assay was 0.32±0.03 μM.    -   i. Treatment with ALD-5 or ALD-2 from 0.1 to 300 μM did not        produce statistically significant neuroprotection from hydrogen        peroxide treatment alone as assessed with CFDA assay.    -   j. Treatment with ALD-5 or ALD-2 from 0.1 to 300 μM did not        produce statistically significant protection from cell death        produced by hydrogen peroxide treatment as assessed with the PI        assay.    -   k. The toxic signal produced by 10 μM hydrogen peroxide was        typical of a wide variety of oxidative stressors (ethanol, heavy        metals, ammonium acetate, and glutamate) that have been tested        in the past, with decreases from control ranging from 30 to 50%.    -   l. The positive control (10 μM cannabidiol) was active on every        test plate, indicating that the model system was responding in a        typical protective manner.

Example 14: In Vivo Pharmacology of NS2-D6 (Compound I-1)

NS2-D6 (Compound ID 100029054-1; Batch Number 1603356191) was tested inbinding and enzyme uptake assays. Compound binding was calculated as a %inhibition of the binding of a radioactively labeled ligand specific foreach target. Compound enzyme inhibition effect was calculated as a %inhibition of control enzyme activity. Results showing an inhibition orstimulation higher than 50% are considered to represent significanteffects of the test compounds. Such effects were observed here and arelisted in the following tables.

Reference Compounds

In each experiment and if applicable, the respective reference compoundwas tested concurrently with NS2-D6, and the data were compared withhistorical values determined at the same research facility. Theexperiment was conducted in accordance with industry standard operatingprocedures.

Results

Table 28 summarizes the enzyme inhibition results.

TABLE 28 Summary of Enzyme Inhibition Results Assay 1.0E−05M5-HT2B^((h)) (agonist radioligand) 62.5% acetylcholinesterase (h) 52.9%MAO-A (antagonist radioligand) 68.5% MT₃ (ML₂) (agonist radioligand)71.1% PR (h) (agonist radioligand) 60.9%

Test compound results are shown in FIGS. 44-46. Table 29 shows specificbinding results for NS2-D6.

TABLE 29 Test Compound Results Client Compound Test % Inhibition ofControl Specific Binding Compound I.D. I.D. Concentration 1^(st) 2^(nd)Mean A₁(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.5 5.3 4.4A_(2A (h)) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −11.2 −15.0−13.1 A_(2B)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M13.2 4.3 8.7 A₃(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M17.3 15.1 16.2 α_(1A)(h) (antagonist radioligand) 100029054-1 NS2-d61.0E−05M 6.1 −3.3 1.4 α_(1B)(h) (antagonist radioligand) 100029054-1NS2-d6 1.0E−05M −1.8 1.0 −0.4 α_(2A)(h) (antagonist radioligand)100029054-1 NS2-d6 1.0E−05M 3.2 10.0 6.6 α_(2B)(h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 15.9 20.8 18.4 α_(2C)(h)(antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −8.0 6.2 −0.9 β₁(h)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 9.7 11.0 10.3 β₂(h)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −0.6 5.8 2.6Adrenergic beta3 100029054-1 NS2-d6 1.0E−05M 16.2 3.0 9.6 AT₁(h)(antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 10.7 −3.3 3.7AT₂(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.5 −5.6 −4.6APJ (apelin)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 0.82.2 1.5 BZD (central) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M−17.2 −16.6 −16.9 BB₃(h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −17.1 4.6 −6.3 B₂(h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −4.5 0.1 −2.2 CB₁(h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M 21.9 20.7 21.3 CB₂(h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M 16.6 5.0 10.8 CCK₁ (CCKA) (h) (agonist radioligand) 100029054-1NS2-d6 1.0E−05M −10.0 11.9 0.9 CCK₂ (CCKB) (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M −3.6 −14.6 −9.1 CRF₁(h) (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M 6.7 5.7 6.2 D₁(h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 0.8 6.6 3.7 D_(2S)(h) (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M 15.2 14.4 14.8 D₃(h)(antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 6.2 −4.1 1.1ET_(A)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −0.8 −5.8−3.3 ET_(B)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 7.6−5.4 1.1 GAB_(A1)(h) (α1 β2, y2)(agonist radioligand) 100029054-1 NS2-d61.0E−05M −45.9 −21.1 −33.5 GABA_(B(1b))(h) (antagonist radioligand)100029054-1 NS2-d6 1.0E−05M −7.2 5.2 −1.0 glucagon(h) (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M −4.2 −6.9 −5.6 AMPA (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M −5.9 5.1 −0.4 kainate (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M 17.5 12.1 14.8 NMDA (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 5.5 14.6 10.1 glycine(strychnine-insenstive) (antagonist radioligand) 100029054-1 NS2-d61.0E−05M −7.7 16.2 4.2 TNF-α (h) (agonist radioligand) 100029054-1NS2-d6 1.0E−05M −6.5 −0.9 −3.7 CCR2 (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M −12.4 6.2 −3.1 H₁(h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M −8.6 19.9 5.6 H₂(h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 1.1 −0.5 0.3 H₃(h) (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M 15.3 13.9 14.6 H₄(h) (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M 9.8 −3.3 3.2 BLT₁ (LTB₄₎(h)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 9.6 7.0 8.3CysLT₁(LTD₄₎(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.6−17.1 −10.4 MCH₁ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M−3.7 −9.3 −6.5 MC₁ (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M−10.5 −5.7 −8.1 MC₃ (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −1.4 0.6 −0.4 MC₄ (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −2.8 −3.0 −2.9 MT₁(ML_(1A))(h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M 30.0 37.7 33.9 MT₃ (ML₂)(h) (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M 69.0 73.3 71.1 MAO-A(antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 66.4 70.7 68.5motilin (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −12.2−3.9 −8.0 M₁ (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M−2.6 0.5 −1.1 M₂ (h) (antagonist radioligand) 100029054-1 NS2-d61.0E−05M −4.2 −0.4 −2.3 M₃ (h) (antagonist radioligand) 100029054-1NS2-d6 1.0E−05M 4.6 13.6 9.1 M₄ (h) (antagonist radioligand) 100029054-1NS2-d6 1.0E−05M −1.0 9.2 4.1 NK₁ (h) (agonist radioligand) 100029054-1NS2-d6 1.0E−05M 4.2 4.1 4.2 NK₂ (h) (agonist radioligand) 100029054-1NS2-d6 1.0E−05M 18.6 −4.5 7.0 Y₁ (h) (agonist radioligand) 100029054-1NS2-d6 1.0E−05M −8.7 7.5 −0.6 N neuronal α4β2 (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M −5.7 −6.7 −6.2 N muscle-type (h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M −16.8 0.7 −8.1 δ (DOP)(h)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.8 −1.4 1.2 κ (KOP)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 13.3 16.2 14.8 μ(MOP) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.0 6.8 4.9NOP (ORLI) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 10.818.7 14.8 PPARy (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M13.1 23.6 18.3 PAF (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M−10.1 7.4 −1.3 PCP (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M2.1 6.5 4.3 EP₂(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M18.1 41.5 29.8 FP (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M10.7 −4.5 3.1 IP (PGI₂) (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M 5.0 −9.1 −2.0 LXRβ (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −10.5 −3.9 −7.2 5-HT_(1A) (h) (agonist radioligand) 100029054-1NS2-d6 1.0E−05M 5.4 −1.8 1.8 5-HT_(1B) (h) (antagonist radioligand)100029054-1 NS2-d6 1.0E−05M −16.3 0.5 −7.9 5-HT_(1D) (h) (agonistradioligand) 100029054-1 NS2-d6 1.0E−05M −3.4 −5.1 −4.2 5-HT_(2A) (h)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 5.4 17.8 11.65-HT_(2B) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 60.864.2 62.5 5-HT_(2C) (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −3.1 10.0 3.4 5-HT₃ (h) (antagonist radioligand) 100029054-1NS2-d6 1.0E−05M −3.0 2.2 −0.4 5-HT_(4e) (h) (antagonist radioligand)100029054-1 NS2-d6 1.0E−05M −5.5 −2.6 −4.1 5-HT_(5a) (h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 3.8 17.3 10.6 5-HT₆ (h)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 1.1 17.7 9.4 5-HT₇ (h)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 2.3 −5.6 −1.7 sigma(non-selective) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M16.3 13.5 14.9 sst₁ (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −5.1 5.0 −0.1 sst₄ (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M 3.1 −5.9 −1.4 GR (h) (agonist radioligand) 100029054-1 NS2-d61.0E−05M −6.7 4.1 −1.3 Estrogen ER alpha (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M 15.8 4.3 10.1 PR (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M 58.7 63.1 60.9 AR (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M 41.4 44.3 42.8 Thyroid Hormone 100029054-1NS2-d6 1.0E−05M −7.9 −5.9 −6.9 UT(h) (agonist radioligand) 100029054-1NS2-d6 1.0E−05M 3.1 4.9 4.0 VPAC₁ (VIP₁) (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M 0.2 1.9 1.1 V_(1a) (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M 2.1 −1.2 0.5 V₂ (h) (agonist radioligand)100029054-1 NS2-d6 1.0E−05M 0.4 2.9 1.7 Ca²⁺ channel (L. dihydropyridinesite) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 5.9 3.8 4.9Ca²⁺ channel (L. diltiazem site) (benzothiazepines) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 14.8 13.3 14.0 Ca²⁺ channel (L.verapamil site) (phenylalkylamine) (antagonist radioligand) 100029054-1NS2-d6 1.0E−05M −6.5 −11.8 −9.1 Ca²⁺ channel (N) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 4.6 8.1 6.3 Potassium ChannelhERG (human)- [3H] Dofetilide 100029054-1 NS2-d6 1.0E−05M 8.0 −2.8 2.6SK_(Ca) channel (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M−10.8 −4.5 −7.7 Na⁺ channel (site 2) (antagonist radioligand)100029054-1 NS2-d6 1.0E−05M −2.7 6.2 1.8 Cl⁻ channel (GABA-gated)(antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 33.9 42.6 38.2norepinephrine transporter (h) (antagonist radioligand) 100029054-1NS2-d6 1.0E−05M 33.9 29.1 31.5 dopamine transporter (h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 19.9 27.3 23.6 GABA transporter(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −6.1 −4.5 −5.3choline transporter (CHT1)(h) (antagonist radioligand) 100029054-1NS2-d6 1.0E−05M −17.9 −13.1 −15.5 5-HT transporter (h) (antagonistradioligand) 100029054-1 NS2-d6 1.0E−05M 2.9 5.2 4.0

Table 30 shows IC₅₀, and nH values for various reference compounds thatwere compared with NS2-D6.

TABLE 30 Reference Compound Results Compound I.D. IC₅₀ (M) K_(i) (M) nHA₁ (h)(agonist radioligand) CPA 3.3E−09M 1.3E−09M 1.2 A_(2A) (h)(agonist radioligand) NECA 3.7E−08M 3.1E−08M 0.8 A_(2B) (h) (antagonistradioligand) NECA 5.5E−07M 5.1E−07M 0.9 A₃ (h) (agonist radioligand)IB-MECA 2.8E−10M 1.6E−10M 0.8 α_(1A) (h) (antagonist radioligand) WS4101 2.5E−10M 1.2E−10M 1.1 α_(1B) (h) (antagonist radioligand) prazosin2.5E−10M 6.7E−11M 1.2 α_(2A) (h) (antagonist radioligand) yohimbine7.5E−09M 3.3E−09M 1.3 α_(2B) (h) (antagonist radioligand) yohimbine7.6E−09M 5.0E−09M 1.0 α_(2C) (h) (antagonist radioligand) yohimbine4.3E−09M 1.4E−09M 1.3 β1 (h) (agonist radioligand) atenolol 3.1E−07M1.7E−07M 0.9 β2 (h) (agonist radioligand) ICI 118551 1.3E−09M 4.5E−10M1.8 Adrenergic beta3 Alprenolo 1 1.4E−07M 1.0E−07M 0.6 AT₁ (h)(antagonist radioligand) saralasin 1.4E−09M 6.9E−10M 1.1 AT₂ (h)(agonist radioligand) angiotensin-II 1.2E−10M 5.9E−11M 0.8 APJ (apelin)(h) (agonist radioligand) apelin-13.TFA 2.7E−10M 2.5E−10M 1.0 BZD(central) (agonist radioligand) diazepam 7.4E−09M 6.2E−09M 0.9 BB₃ (h)(agonist radioligand) Bn(6-14) 9.8E−09M 6.0E−09M 1.0 B₂ (h) (agonistradioligand) NPC 567 2.4E−08M 1.2E−08M 1.0 CB₁ (h) (agonist radioligand)CP 55940 1.1E−09M 9.5E−10M 1.2 CB2 (h) (agonist radioligand) WIN 55212-22.1E−09M 1.4E−09M 1.0 CCK₁ (CCK_(A)) (h)(agonist radioligand) CCK-8s1.1E−10M 8.3E−11M 0.9 CCK₂(CCK_(B)) (h)(agonist radioligand) CCK-8s1.1E−10M 4.6E−11M 1.0 CRF₁ (h) (agonist radioligand) sauvagine 5.5E−10M3.4E−10M 1.0 D₁ (h) (antagonist radioligand) SCH 23390 2.1E−10M 8.4E−11M0.7 D_(2S) (h) (agonist radioligand) 7-OH-DPAT 1.2E−09M 4.8E−10M 0.7D₃(h) (antagonist radioligand) (*) butaclamol 1.5E−09M 3.2E−10M 1.0ET_(A) (h) (agonist radioligand) endothelin-1 5.4E−11M 2.7E−11M 1.0ET_(B) (h) (agonist radioligand) endothelin-3 2.5E−11M 1.4E−11M 0.9GABA_(A1) (h) (α1,(β2, γ2) (agonist radioligand) muscimol 7.0E−08M4.7E−08M 0.8 GABA_(B(1b)) (h) (antagonist radioligand) CGP 546262.9E−09M 1.5E−09M 0.7 glucagon (h) (agonist radioligand) glucagon1.5E−09M 1.1E−09M 0.6 AMPA (agonist radioligand) L-glutamate 2.8E−07M2.6E−07M 0.9 kainate (agonist radioligand) kainic acid 2.3E−08M 1.8E−08M0.9 NMDA (antagonist radioligand) CGS 19755 2.1E−07M 1.7E−07M 0.9glycine (strychnine-insensitive) (antagonist radioligand) glycine1.4E−07M 1.3E−07M 1.0 TNF-α (h) (agonist radioligand) TNF-alpha 6.9E−11M2.3E−11M 1.3 CCR2 (h) (agonist radioligand) MCP-1 2.9E−11M 1.2E−11M 1.6H₁ (h) (antagonist radioligand) pyrilamine 2.5E−09M 1.6E−09M 1.3 H₂ (h)(antagonist radioligand) cimetidine 6.5E−07M 6.3E−07M 0.8 H₃ (h)(agonist radioligand) (R)a-Me-histamine 1.9E−09M 4.7E−10M 1.4 H₄ (h)(agonist radioligand) imetit 4.4E−09M 1.9E−09M 0.9 BLT₁ (LTD₄) (h)(agonist radioligand) LTB₄ 4.2E−10M 2.1E−10M 0.8 CysLT₁ (LTD₄) (h)(agonist radioligand) LTD₄ 7.0E−10M 3.1E−10M 1.0 MCH₁ (h) (agonistradioligand) human MCH 4.9E−11M 4.5E−11M 1.0 MCI (agonist radioligand)NDP-α-MSH 1.8E−10M 8.9E−11M 1.0 MC3 (h) (agonist radioligand) NDP-α-MSH2.0E−10M 1.7E−10M 1.2 MC₁ (h) (agonist radioligand) NDP-α-MSH 4.8E−10M4.4E−10M 0.7 MT₁ (ML_(1A)) (h) (agonist radioligand) melatonin 2.0E−10M1.6E−10M 1.6 MT₃ (ML₂) (agonist radioligand) melatonin 7.2E−08M 7.1E−08M0.8 MAO-A (antagonist radioligand) clorgyline 1.7E−09M 1.0E−09M 1.7motilin (h) (agonist radioligand) [Nleu¹³]-motilin 2.0E−09M 1.7E−09M 1.1M₁ (h) (antagonist radioligand) pirenzepine 2.2E−08M 1.9E−08M 1.0 M₂ (h)(antagonist radioligand) methoctramine 4.9E−08M 3.4E−08M 0.8 M₃ (h)(antagonist radioligand) 4-DAMP 1.8E−09M 1.3E−09M 1.3 M₄ (h) (antagonistradioligand) 4-DAMP 1.5E−09M 9.5E−10M 1.2 NK₁ (h) (agonist radioligand)(Sar⁹,Met(O₂)¹¹]-SP 4.9E−10M 2.2E−10M 1.6 NK₂ (h) (agonist radioligand)[Nleu¹⁰]-NKA (4-10) 3.1E−09M 1.7E−09M 0.8 Y₁ (h) (agonist radioligand)NPY 9.4E−11M 6.7E−11M 1.3 N neuronal α4β2 (h) (agonist radioligand)nicotine 4.5E−09M 1.5E−09M 0.9 N muscle-type (h) (antagonistradioligand) α-bungarotoxin 2.3E−09M 2.1E−09M 1.2 δ (DOP) (h) (agonistradioligand) DPDPE 2.7E−09M 1.6E−09M 0.9 κ (KOP) (agonist radioligand)U50488 9.3E−10M 6.2E−10M 1.0 μ (MOP) (h) (agonist radioligand) DAMGO2.8E−10M 1.2E−10M 0.7 NOP (ORLI) (h) (agonist radioligand) nociceptin8.4E−10M 1.1E−10M 1.1 PPAR_(γ) (h) (agonist radioligand) rosiglitazone1.2E−08M 6.1E−09M 0.9 PAF (h) (agonist radioligand) C₁₆-PAF 5.8E−09M2.9E−09M 1.8 PCP (antagonist radioligand) MK 801 9.2E−09M 5.2E−09M 1.3EP₂ (h) (agonist radioligand) PGE2 3.4E−09M 1.7E−09M 1.1 FP (h) (agonistradioligand) PGF2alpha 1.9E−09M 1.2E−09M 0.9 IP (PGI₂) (h) (agonistradioligand) iloprost 1.8E−08M 1.0E−08M 0.9 LXRβ (h) (agonistradioligand) 22(R)-hydroxycholesterol 4.0E−06M 2.7E−06M 1.1 5-HT_(1A)(h) (agonist radioligand) 8-OH-DPAT 5.8E−10M 3.6E−10M 0.8 5-HT_(1B)(antagonist radioligand) serotonin 4.9E−09M 3.0E−09M 0.9 5-HT_(1D)(agonist radioligand) serotonin 2.4E−09M 8.1E−10M 1.2 5-HT_(2A) (h)(agonist radioligand) (±)DO1 3.4E−10M 2.5E−10M 0.7 5-HT_(2B) (h)(agonist radioligand) (±)DO1 6.9E−09M 3.4E−09M 0.9 5-HT_(2C) (h)(agonist radioligand) (±)DO1 5.3E−10M 4.7E−10M 1.1 5-HT₃ (h) (antagonistradioligand) MDL 72222 6.9E−09M 4.8E−09M 0.9 5-HT_(4e) (h) (antagonistradioligand) serotonin 2.7E−07M 8.9E−08M 0.7 5-HT_(5α) (h) (agonistradioligand) serotonin 1.5E−07M 7.5E−08M 1.0 5-HT₆ (h) (agonistradioligand) serotonin 2.0E−07M 9.3E−08M 1.1 5-HT₇ (h) (agonistradioligand) serotonin 3.4E−10M 1.3E−10M 1.0 sigma (non-selective) (h)(agonist radioligand) haloperidol 6.3E−08M 5.1E−08M 0.7 sst₁ (h)(agonist radioligand) somatostatin-28 2.0E−10M 1.9E−10M 0.8 sst₄ (h)(agonist radioligand) somatostatin-14 9.1E−10M 8.9E−10M 0.9 GR (h)(agonist radioligand) dexamethasone 4.9E−09M 2.4E−09M 1.2 Estrogen ERalpha (h) (agonist radioligand) Diethylstilbestrol 3.7E−10M 1.0E−10M 1.9PR (h) (agonist radioligand) promegestone 4.7E−10M 3.8E−10M 1.6 AR (h)(agonist radioligand) mibolerone 1.6E−09M 6.9E−10M 1.2 Thyroid HormoneTriiodothyronine 4.2E−11M 2.3E−11M 1.1 UT (h) (agonist radioligand)urotensin-II 7.7E−10M 5.8E−10M 1.1 VPAC₁ (VIP₁) (h) (agonistradioligand) VIP 3.5E−10M 1.9E−10M 1.9 V_(1a) (h) (agonist radioligand)[d(CH₂)₅ ¹, Tyr(ME)₂]-AVP 1.4E−09M 8.8E−10M 1.0 V₂ (h) (agonistradioligand) AVP 4.3E−10M 3.1E−10M 0.7 Ca²⁺ channel (L. dihydropyridinesite) (antagonist radioligand) nitrendipine 3.0E−10M 1.9E−10M 1.1 Ca²⁺channel (L. diltiazem site) (benzothiazepines) (antagonist radioligand)diltiazem 6.8E−08M 5.3E−08M 1.1 Ca²⁺ channel (L. verapamil site)(phenylalkylamine) (antagonist radioligand) D 600 2.7E−08M 1.3E−08M 0.5Ca²⁺ channel (N) (antagonist radioligand) ω-conotoxin GVIA 1.7E−12M6.8E−13M 0.8 Potassium Channel hERG (human)- [3H] Dofetilide Terfenadine3.4E−08M 2.3E−08M 0.8 SK_(Ca) channel (antagonist radioligand) apamin8.8E−12M 4.4E−12M 1.0 Na⁺ channel (site 2) (antagonist radioligand)veratridine 3.9E−06M 3.5E−06M 0.8 Cl⁻ channel (GABA-gated) (antagonistradioligand) picrotoxinin 2.8E−07M 2.4E−07M 1.0 norepinephrinetransporter (h) (antagonist radioligand) protriptyline 2.8E−09M 2.1E−09M1.0 dopamine transporter (h) (antagonist radioligand) BTCP 9.5E−09M5.0E−09M 1.1 GABA transporter (h) (antagonist radioligand) nipecoticacid 2.5E−06M 2.5E−06M 0.8 choline transporter (CHT1)(h) (antagonistradioligand) hemicholinium-3 7.5E−09M 4.2E−09M 1.0 5-HT transporter (h)(antagonist radioligand) imipramine 2.7E−09M 1.3E−09M 1.3

FIG. 47 shows a histogram of in vitro pharmacology results in enzyme anduptake assays for NS2-D6.

Table 31 shows % inhibition of control values for NS2-D6.

TABLE 31 Test Compound Results Client Test % inhibition of ControlValues Flags Compound I.D. Compound I.D. Concentration 1^(st) 2^(nd)Mean 1^(st) 2^(nd) COX1(h) 100029054-1 NS2-d6 1.0E−05 M 20.1 −2.6 8.8COX2(h) 100029054-1 NS2-d6 1.0E−05 M 12.8 3.0 7.9 5-lipoxygenase (h)100029054-1 NS2-d6 1.0E−05 M 23.5 24.8 24.2 12-lipoxygenase (h)100029054-1 NS2-d6 1.0E−05 M −9.4 −4.1 −6.7 inducible NOS 100029054-1NS2-d6 1.0E−05 M 2.8 −2.0 0.4 PDE2A1 (h) 100029054-1 NS2-d6 1.0E−05 M17.1 −24.5 −3.7 PDE3B (h) 100029054-1 NS2-d6 1.0E−05 M 0.9 −4.5 −1.8PDE4D2 (h) 100029054-1 NS2-d6 1.0E−05 M 15.9 16.9 16.4 PDE5 (h)(non-selective) 100029054.1 NS2-d6 1.0E−05 M −8.6 −2.9 −5.7 PDE6(non-selective) 100029054.1 NS2-d6 1.0E−05 M 4.1 18.9 11.5 ACE (h)100029054.1 NS2-d6 1.0E−05 M 127.6 131.0 129.3 INTER INTER ACE-2 (h)100029054-1 NS2-d6 1.0E−05 M 23.8 26.2 25.0 INTER INTER BACE-1 (h)(β-secretase) 100029054-1 NS2-d6 1.0E−05 M −4.1 −5.7 −4.9 INTER INTERcaspase-3 (h) 100029054-1 NS2-d6 1.0E−05 M −2.0 2.5 0.2 caspase-8 (h)100029054-1 NS2-d6 1.0E−05 M 14.7 16.4 15.6 HIV-1 protease 100029054-1NS2-d6 1.0E−05 M 16.6 4.8 10.7 MMP-1 (h) 100029054-1 NS2-d6 1.0E−05 M45.3 10.7 28.0 INTER INTER MMP-2 (h) 100029054-1 NS2-d6 1.0E−05 M 4.9−2.4 1.3 MMP-9 (h) 100029054-1 NS2-d6 1.0E−05 M 4.5 4.4 4.4 INTER INTERAbl kinase (h) 100029054-1 NS2-d6 1.0E−05 M −4.8 −11.6 −8.2 CaMK2α (h)100029054-1 NS2-d6 1.0E−05 M 0.2 −6.5 −3.1 CDK2 (h) (cycA) 100029054-1NS2-d6 1.0E−05 M 1.2 −5.1 −1.9 ERK₂ (h) (P42^(mapk)) 100029054-1 NS2-d61.0E−05 M −6.3 −1.3 −3.8 FLT-1 kinase (h) (VEGFR1) 100029054-1 NS2-d61.0E−05 M −15.5 −18.8 −17.2 Fyn kinase (h) 100029054-1 NS2-d6 1.0E−05 M−8.6 −0.3 −4.5 IRK (h)(InsR) 100029054-1 NS2-d6 1.0E−05 M −15.1 −7.9−11.5 Lyn A kinase (h) 100029054-1 NS2-d6 1.0E−05 M −17.7 −35.7 −26.7p38α kinase (h) 100029054-1 NS2-d6 1.0E−05 M −3.2 −3.1 −3.2 ZAP70 kinase(h) 100029054-1 NS2-d6 1.0E−05 M 0.4 1.5 1.0 acetylcholinesterase (h)100029054-1 NS2-d6 1.0E−05 M 45.8 60.1 52.9 COMT (catechol-O-methyltransferase) 100029054-1 NS2-d6 1.0E−05 M 5.8 14.7 10.3 INTER INTERMAO-B (h) recombinant enzyme 100029054-1 NS2-d6 1.0E−05 M −3.1 −3.7 −3.4xanthine oxldase/superoxide 0₂ ⁻scavenging 100029054-1 NS2-d6 1.0E−05 M−3.1 15.6 6.3 ATPase (Na+/K+) 100029054-1 NS2-d6 1.0E−05 M 5.2 −0.4 2.4Peptidase, Metalloproteinase, Neutral Endopeptidase 100029054-1 NS2-d61.0E−05 M 0.3 3.0 1.7 INTER: Test compound interferes with the assaydetection method.

Table 32 shows IC₅₀ and nH values for reference compounds.

TABLE 32 Reference Compound IC₅₀ and nH Values Compound I.D. IC₅₀ (IV)nH COX1(h) Diclofenac 5.7E−09M 1.6 COX2(h) NS398 6.8E−08M 1.35-lipoxygenase (h) NDGA 2.3E−07M 1.6 12-lipoxygenase (h) NDGA 5.1E−07M1.2 inducible NOS 1400W 4.0E−08M 1.4 PDE2A1 (h) EHNA 1.1E−06M 0.8 PDE3B(h) milrinone 1.0E−06M 0.9 PDE4D2 (h) Ro 20-1724 8.2E−07M 1.0 PDE5 (h)(non-selective) dipyridamole 1.8E−06M 1.1 PDE6 (non-selective) zaprinast1.8E−07M 1.0 ACE (h) captopril 5.7E−10M 1.2 ACE-2 (h) Ac-GG-26-NH₂2.9E−07M 2.4 BACE-1 (h) ((β-secretase) OM 99-2 1.2E−07M 1.4 caspase-3(h) Ac-DEVD-CHO 2.0E−09M 1.1 caspase-8 (h) Ac-lETD-CHO 2.8E−08M 0.8HIV-1 protease pepstatin A 2.2E−06M 1.9 MMP-1 (h) GM6001 1.6E−09M 1.3MMP-2 (h) GM6001 1.5E−09M 1.3 MMP-9 (h) GM6001 5.4E−10M 0.9 Abl kinase(h) staurosporine 2.6E−07M 1.4 CaMK2u (h) AIP 2.6E−07M 1.1 CDK2 (h)(cycA) staurosporine 6.9E−09M 1.0 ERK2 (0) (P42^(mapk)) staurosporine6.9E−07M 1.0 FLT-1 kinase (h) (VEGFR1) staurosporine 7.0E−09M 0.7 Fynkinase (h) PP1 1.0E−07M >3 IRK (h) (InsR) staurosporine 1.6E−08M 0.9 LynA kinase (h) staurosporine 1.2E−08M 1.9 p38α kinase (h) SB2021903.0E−08M 1.0 ZAP70 kinase (h) staurosporine 1.1E−07M 1.4acetylcholinesterase (h) galanthamine 7.6E−07M 1.0 COMT(catechol-O-methyl transferase) Ro 41-0960 3.0E−08M 1.7 MAO-B (h)recombinant enzyme deprenyl 3.5E−08M 1.4 xanthine oxidase/superoxide0₂-scavenging allopurinol 2.0E−06M 1.3 ATPase (Na+/K+) ouabain 1.0E−06M1.3 Peptidase, Metalloproteinase, Neutral Endopeptidase Phosphoramidon1.6E−08M 0.9

Table 33 shows test compound results of NS2-D6 on guanylyl cyclase.

TABLE 33 Test Compound Results with Guanylyl Cyclase Client Test % ofControl Values Compound I.D. Compound I.D. Concentration 1^(st) 2^(nd)Mean guanylyl cyclase (h) (activator effect) 100029054-1 NS2-d6 1.0E−05M0.2 0.3 0.2

Table 34 shows reference compound EC₅₀ results on guanylyl cyclase.

TABLE 34 Reference Compound Results with Guanylyl Cyclase Compound I.D.EC₅₀(M) nH guanylyl cyclase (h) (activator effect) sodium nitroprusside3.5E−06M 2.2

Results showing an inhibition (or stimulation for assays run in basalconditions) higher than 50% are considered to represent significanteffects of the test compounds. 50% is the most common cut-off value forfurther investigation (determination of IC₅₀ or EC₅₀ values fromconcentration-response curves). Results showing an inhibition (orstimulation) between 25% and 50% are indicative of weak to moderateeffects (in most assays, they should be confirmed by further testing asthey are within a range where more inter-experimental variability canoccur). Results showing an inhibition (or stimulation) lower than 25%are not considered significant and mostly attributable to variability ofthe signal around the control level.

Low to moderate negative values have no real meaning and areattributable to variability of the signal around the control level. Highnegative values (>50%) that are sometimes obtained with highconcentrations of test compounds are generally attributable tononspecific effects of the test compounds in the assays. On rareoccasions they could suggest an allosteric effect of the test compound.

Experimental Conditions

Table 35 summarizes binding assay conditions

TABLE 35 Binding Assay Conditions Detection Assay Source Ligand Conc. KdNon Specific Incubation Method Bibl. A1(h) (agonist human [³H]CCPA     1nM    0.7 nM CPA   60 min Scintillation 198 radioligand) recombinant (10μM) RT counting (CHO cells) A_(2A) (h)(agonist human [³H]CGS 21680     6nM     27 nM NECA  120 min Scintillation 141 radioligand) recombinant(10 μM) RT counting (HEK-293 cells) A_(2B)(h) (antagonist human [³H]CPX    5 nM     65 nM NECA   60 min Scintillation 229 radioligand)recombinant (100 μM) RT counting (HEK-293 cells) A₃(h) (antagonist human[¹²⁵]AB-MECA  0.15 nM   0.22 nM IB-MECA  120 min Scintillation 206radioligand) recombinant (1 μM) RT counting (HEK-293 cells) α_(1A)(h)(antagonist human [³H]prazosin   0.1 nM    0.1 nM epinephrine   60 minScintillation 897 radioligand) recombinant (0.1 μM) RT counting (CHOcells) α_(1B)(h) (antagonist human [³H]prazosin  0.15 nM  0.055 nMphentolamine   60 min Scintillation 701 radioligand) recombinant (10 μM)RT counting (CHO cells) α_(2A)(h) (antagonist human [³H]RX 821002     1nM    0.8 nM (−)epinephrine   60 min Scintillation 542 radioligand)recombinant (100 μM) RT counting (CHO cells) α_(2B)(h) (antagonist human[³H]RX 821002   2.5 nM      5 nM (−)epinephrine   60 min Scintillation56 radioligand) recombinant (100 μM) RT counting (CHO cells) α_(2C)(h)(antagonist human [³H]RX 821002     2 nM   0.95 nM (−)epinephrine   60min Scintillation 56 radioligand) recombinant (100 μM) RT counting (CHOcells) β₁(h) (agonist human [³H](−)CGP   0.3 nM   0.39 nM alprenolol  60 min Scintillation 548 radioligand) recombinant 12177 (50 μM) RTcounting (HEK-293 cells) β₂(h) (agonist human [³H](−)CGP   0.3 nM   0.15nM alprenolol  120 min Scintillation 794 radioligand) recombinant 12177(50 μM) RT counting (CHO cells) Adrenergic beta3 human [125I]   0.5 nM   1.5 nM Alprenolol(100   90 min Scintillation 1277 recombinantCyanopindolol (0.0 μM) 25° C. counting (HEK-293 cells) AT₁(h)(antagonist human [¹²⁵I][Sar¹, Ile⁸]-AT-  0.05 nM   0.05 nMangiotensin-II  120 min Scintillation 776 radioligand) recombinant II(10 μM) 37° C. counting (HEK-293 cells) AT₂(h) (agonist human [¹²⁵I]GCP 0.01 nM   0.01 nM angiotensin-II  4 hr Scintillation 248 radioligand)recombinant 42112A (1 μM) 37° C. counting (HEK-293 cells) APJ(apelin)(h) human [¹²⁵I]  0.03 nM   0.06 nM apelin-13  120 minScintillation 846 (agonist radioligand) recombinant [Glpr⁶⁵, Nle⁷⁵],Tyr⁷⁷- (1 μM) RT counting (CHO cells) apelin-13 BB₃(h) (agonist human[¹²⁵I]Bn(6-14)  0.01 nM   0.16 nM Bn (6-14)   60 min Scintillation 287radioligand) recombinant (1 μM) RT counting (CHO cells) B₂(h) (agonisthuman [³H]bradykinin   0.3 nM   0.32 nM bradykinin   60 minScintillation 346 radioligand) recombinant (1 μM) RT counting (CHOcells) CB₁(h) (agonist human [³H](−)CP 55940   0.5 nM    3.5 nM WIN55212-2  120 min Scintillation 857 radioligand) recombinant (10 μM) 37°C. counting (CHO cells) CB₂(h) (agonist human [³H]WIN   0.8 nM    1.5 nMWIN 55212-2  120 min Scintillation 165 radioligand) recombinant 55212-2(5 μM) 37° C. counting (CHO cells) CCK₁ (CCK_(A)) (h) human [¹²⁵I]CCK-8s 0.08 nM   0.24 nM CCK-8s   60 min Scintillation 562 (agonistradioligand) recombinant (1 μM) RT counting (CHO cells) CCK₂ (CCK_(B))(h) human [¹²⁵I]CCK-8s  0.08 nM  0.054 nM CCK-8s   60 min Scintillation134 (agonist radioligand) recombinant (1 μM) RT counting (CHO cells)CRF₁(h) (agonist human [¹²⁵I]sauvagine 0.075 nM   0.12 nM sauvagine  120min Scintillation 557 radioligand) recombinant (0.5 μM) RT counting (CHOcells) D₁(h) (antagonist human [³H]SCH 23390   0.3 nM    0.2 nM SCH23390   60 min Scintillation 281 radioligand) recombinant (1 μM) RTcounting (CHO cells) D_(2S)(h) (agonist human [³H]7-OH-DPAT     1 nM  0.68 nM butaclamol   60 min Scintillation 87 radioligand) recombinant(10 μM) RT counting (HEK-293 cells) D₃(h) (antagonist human [³H]methyl-  0.3 nM  0.085 nM (+)butaclamol   60 min Scintillation 145 radioligand)recombinant spiperone (10 μM) RT counting (CHO cells) ET_(A)(h) (agonisthuman [¹²⁵I]endothelin-1  0.03 nM   0.03 nM endothelin-1  120 minScintillation 30 radioligand) recombinant (100 μM) 37° C. counting (CHOcells) ET_(B)(h) (agonist human [¹²⁵I]endothelin-1  0.03 nM   0.04 nMendothelin-1  120 min Scintillation 541 radioligand) recombinant (0.1μM) 37° C. counting (CHO cells) GAB_(A1)(h) (α1 β2, human [³H]muscimol   15 nM     30 nM muscimol  120 min Scintillation 109 y2)(agonistrecombinant (10 μM) RT counting radioligand) (CHO cells) GABA_(B(1b))(h)human [³H]CGP     1 nM      1 nM CGP 52432  120 min Scintillation 508(antagonist recombinant 54626 (100 μM) RT counting radioligand) (CHOcells) glucagon(h) (agonist human [¹²⁵I]glucagon 0.025 nM  0.069 nMglucagon  120 min Scintillation 624 radioligand) recombinant (1 μM) RTcounting (CHO cells) TNF-α (h) (agonist U-937 cells [¹²⁵I]TNF-α   0.1 nM  0.05 nM TNF-α  120 min Scintillation 26 radioligand) (10 μM)  4° C.counting CCR2 (h) (agonist human [¹²⁵I]MCP-1  0.01 nM  0.007 nM MCP-1  60 min Scintillation 13 radioligand) recombinant (10 nM) RT counting(HEK-293 cells) H₁(h) (antagonist human [³H]pyrilamine     1 nM    1.7nM pyrilamine   60 min Scintillation 492 radioligand) recombinant (1 μM)RT counting (HEK-293 cells) H₂(h) (antagonist human [¹²⁵I]APT 0.075 nM   2.9 nM tiotidine  120 min Scintillation 540 radioligand) recombinant(100 μM) RT counting (CHO cells) H₃(h) (agonist human [³H]N^(α)-Me-    1 nM   0.32 nM (R)α-Me-   60 min Scintillation 563 radioligand)recombinant histamine histamine RT counting (CHO cells) (1 μM) H₄(h)(agonist human [³H]histamine    10 nM    7.6 nM imetit   60 minScintillation 631 radioligand) recombinant (1 μM) RT counting (HEK-293cells) BLT₁ (LTB₄₎(h) human [³H]LTB₄   0.2 nM    0.2 nM LTB₄   60 minScintillation 616 (agonist radioligand) recombinant (0.2 μM) RT counting(CHO cells) CysLT₁ (LTD₄)(h) human [³H]LTD₄   0.3 nM   0.24 nM LTD₄   60min Scintillation 618 (agonist radioligand) recombinant (1 μM) RTcounting (CHO cells) MCH₁ (h) (agonist human [¹²⁵I]Phe¹³, Tyr¹⁹-   0.1nM      1 nM human MCH   60 min Scintillation 526 radioligand)recombinant MCH (0.1 μM) RT counting (CHO cells) MC₁ (agonist B-16-F1cells [¹²⁵I]NDP-α-MSH  0.05 nM   0.05 nM NDP-α-MSH   90 minScintillation 390 radioligand) (endogenous) (1 μM) RT counting MC₃ (h)(agonist human [¹²⁵I]NDP-α-MSH 0.075 nM    0.4 nM NDP-α-MSH   60 minScintillation 211 radioligand) recombinant (1 μM) 37° C. counting (CHOcells) MC₄ (h) (agonist human [¹²⁵I]NDP-α-MSH  0.05 nM   0.54 nMNDP-α-MSH  120 min Scintillation 211 radioligand) recombinant (1 μM) 37°C. counting (CHO cells) MT₁ (ML_(1A))(h) human [¹²⁵I]2-  0.01 nM   0.04nM melatonin   60 min Scintillation 639 (agonist radioligand)recombinant iodomelatonin (1 μM) RT counting (CHO cells) MT₃ (ML₂)(h)Hamster brain [¹²⁵I]2-   0.1 nM    4.8 nM melatonin   60 minScintillation 186 (agonist radioligand) iodomelatonin (30 μM)  4° C.counting motillin (h) human [¹²⁵I]motilin  0.05 nM   0.26 nM[Nleu¹³]-motilin  120 min Scintillation 285 (antagonist recombinant (1μM) RT counting radioligand) (CHO cells) M₁ (h) (antagonist human[³H]pirenzepine     2 nM     13 nM atropine   60 min Scintillation 59radioligand) recombinant (1 μM) RT counting (CHO cells) M₂ (h)(antagonist human [³H]AF-DX 384     2 nM    4.6 nM atropine   60 minScintillation 59 radioligand) recombinant (1 μM) RT counting (CHO cells)M₃ (h) (antagonist human [³H]4-DAMP   0.2 nM    0.5 nM atropine   60 minScintillation 546 radioligand) recombinant (1 μM) RT counting (CHOcells) M₄ (h) (antagonist human [³H]4-DAMP   0.2 nM   0.32 nM atropine  60 min Scintillation 59 radioligand) recombinant (1 μM) RT counting(CHO cells) NK₁ (h) (agonist U373MG [¹²⁵I]-Substance P  0.05 nM   0.04nM [Sar⁹,   30 min Scintillation 104 radioligand) uppsala LYS3Met(O₂)¹¹]-SP RT counting (1 μM) NK₂ (h) (agonist human [¹²⁵I]NKA   0.1nM   0.12 nM [Nieu ¹⁰]-NKA   60 min Scintillation 3 radioligand)recombinant (4-10) RT counting (CHO cells) (300 μM) Y₁ (h) (agonistSK-N-MC [¹²⁵I]peptide YY 0.025 nM   0.06 nM NPY  120 min Scintillation391 radioligand) cells (1 μM) 37° C. counting (endogenous) N neuronalα4β2 (h) SH-SY5Y [³H]cytisine   0.6 nM    0.3 nM nicotine  120 minScintillation 1084 (agonist radioligand) cells (human (10 μM)  4° C.counting recombinant) N muscle-type (h) TE671 cells [¹²⁵I]α-   0.5 nM     5 nM a-bungarotoxin  120 min Scintillation 524 (antagonist(endogenous) bungarotoxin (5 μM) RT counting radioligand) ō (DOP) (h)(agonist human [³H]DADLE   0.5 nM   0.73 nM naltrexone  120 minScintillation 501 radioligand) recombinant (10 μM) RT counting (CHOcells) κ (KOP) (h) (agonist rat [³H]U 69593     1 nM      2 nM naloxone  60 min Scintillation 771 radioligand) recombinant (10 μM) RT counting(CHO cells) μ (MOP) (h) (agonist human [³H]DAMGO   0.5 nM   0.35 nMnaloxone  120 min Scintillation 260 radioligand) recombinant (10 μM) RTcounting (HEK-293 cells) NOP (ORL1) (h) human [³H]nociceptin   0.2 nM   0.4 nM nociceptin   60 min Scintillation 7 (agonist radioligand)recombinant (1 μM) RT counting (HEK-293 cells) PPARy (h) (agonist human[³H]msiglhazone     5 nM    5.7 nM rosiglltazone  120 min Scintillation567 radioligand) recombinant (10 μM)  4° C. counting (E. coli) PAF (h)(agonist human [³H]C₁₆-PAF   1.5 nM    1.5 nM WEB 2086   60 minScintillation 531 radioligand) recombinant (10 μM) RT counting (CHOcells) EP₂ (h) (agonist human [³H]PGE₂     3 nM      3 nM PGE2  120 minScintillation 781 radioligand) recombinant (10 μM) RT counting (HEK-293cells) FP(h) (agonist human [³H]PGF_(2 α)     2 nM   3.83 nMcloprostenol (10   60 min Scintillation 781 radioligand) recombinant μM)RT counting (HEK-293 cells) IP(PGI₂)(h) (agonist human [³H]iloprost    6 nM      8 nM iloprost   60 min Scintillation 781 radioligand)recombinant (10 μM) RT counting (HEK-293 cells) LXRβ(h) (agonist human[³H]hydroxycholesterol    25 nM     55 nM 22(R)-   60 min Scintillation856 radioligand) recombinant hydroxycholesterol RT counting (BL21/DE3(30 μM) cells) 5-HT_(1A) (h) (agonist human [²H)8-OH-   0.3 nM    0.5 nM8-OH-DPAT   60 min Scintillation 164 radioligand) recombinant DPAT (10μM) RT counting (HEK-293 cells) 5-HT_(1B) (h) rat cerebral [¹²⁵I]CYP  0.1 nM   0.16 nM serotonin  120 min Scintillation 111 (antagonistcodex (+30 μM (10 μM) 37° C. counting radioligand) isoproterenol)5-HT_(1D) (h) (agonist rat (³H]serotonin     1 nM    0.5 nM serotonin  60 min Scintillation 777 radioligand) recombinant (10 μM) RT counting(CHO cells) 5-HT_(2A) (h) (agonist human [¹²⁵I](⁺)DOI   0.1 nM    0.2 nM(⁺)DOI   60 min Scintillation 288 radioligand) recombinant (1 μM) RTcounting (HEK-293 cells) 5-HT_(2B) (h) (agonist human [¹²⁵I](⁺)DOI   0.2nM    0.2 nM (⁺)DOI   60 min Scintillation 571 radioligand) recombinant(1 μM) RT counting (CHO cells) 5-HT_(2C) (h) (agonist human [¹²⁵I](⁺)DOI  0.1 nM    0.9 nM (⁺)DOI   60 min Scintillation 288 radioligand)recombinant (10 μM) 37° C. counting (HEK-293 cells) 5-HT_(4e) (h) human[³H]GR 113808   0.3 nM   0.15 nM serotonin   60 min Scintillation 309(antagonist recombinant (100 μM) 37° C. counting radioligand) (CHOcells) 5-HT_(5a) (h) human [³H]LSD   1.5 nM    1.5 nM serotonin  120 minScintillation 193 (antagonist recombinant (100 μM) 37° C. countingradioligand) (HEK-293 cells) 5-HT₆ (h) (agonist human [³H]LSD     2 nM   1.8 nM serotonin  120 min Scintillation 161 radioligand) recombinant(100 μM) 37° C. counting (CHO cells) 5-HT₇ (h) (agonist human [³H]LSD    4 nM    2.3 nM serotonin  120 min Scintillation 217 radioligand)recombinant (10 μM) RT counting (CHO cells) sigma (non-selective) Jurkatcells [3H]DTG    10 nM     41 nM Haloperidol (10  120 min Scintillation1136 (h) (agonist (endogenous) μM) RT counting radioligand) sst₁ (h)(agonist human [¹²⁵I]Tyr¹¹-   0.1 nM      1 nM somatostatin-2  180 minScintillation 761 radioligand) recombinant somatostatin-1 4 8 37° C.counting (CHO cells) (1 μM) sst₄ (h) (agonist human [¹²⁵I]Tyr¹¹-   0.1nM    5.9 nM somatostatin-1  120 min Scintillation 296 radioligand)recombinant somatostatin-1 4 4 RT counting (CHO cells) (1 μM) GR (h)(agonist IM-9 cells [³H]dexamethasone   1.5 nM    1.5 nM triamcinolone 6 hr Scintillation 283 radioligand) (cytosol) (10 μM)  4° C. countingEstrogen ER alpha human [3H]Estradiol   0.5 nM   0.20 nMDiethylstilbestrol  120 min Scintillation 1280 (h) (agonist recombinant(1 μM) RT counting radioligand) (sf9 cells) PR (h) (agonist T47D cells[³H]progesterone   0.5 nM      2 nM promegestone (1 20 hr Scintillation930 radioligand) (cytosol) μM)  4° C. counting AR (h) (agonist LNCaPcells [³H]methyltrienolone     1 nM    0.8 nM mibolerone (1 24 hrScintillation 498 radioligand) (cytosol) μM)  4° C. counting ThyroidHormone rat liver [125I]  0.03 nM  0.034 nM Triodothymnine 1080 minScintillation 1289 Triiodothyronine (1.0 μM)  4° C. counting UT(h)(agonist human [¹²⁵I]urotensin-II   0.1 nM   0.29 nM urotensin-II (3 120 min Scintillation 622 radioligand) recombinant μM) RT counting (CHOcells) VPAC₁ (VIP₁)(h) human [¹²⁵I]VIP  0.04 nM   0.05 nM VIP   60 minScintillation 50 (agonist radioligand) recombinant (1 μM) RT counting(CHO cells) V_(1a) (h) (agonist human [³H]AVP   0.3 nM    0.5 nM AVP  60 min Scintillation 343 radioligand) recombinant (1 μM) RT counting(CHO cells) V₂ (h) (agonist human [³H]AVP   0.3 nM   0.76 nM AVP 120 minScintillation 343 radioligand) recombinant (1 μM) RT counting (CHOcells) Ion channels BZD (central) rat cerebral [³H]flunitrazepam   0.4nM    2.1 nM diazepam   60 min Scintillation 227 (agonist radioligand)cortex (3 μM)  4° C. counting AMPA (agonist rat cerebral [³H]AMPA     8nM     82 nM L-glutamate (1   60 min Scintillation 166 radioligand)cortex mM)  4° C. counting kainate (agonist rat cerebral [³H]kainic acid    5 nM     19 nM L-glutamate (1   60 min Scintillation 160radioligand) cortex mM)  4° C. counting NMDA (antagonist rat cerebral[³H]CGP     5 nM     23 nM L-glutamate   60 min Scintillation 221radioligand) cortex 39653 (100 μM)  4° C. counting glycine (strychnine-rat cerebral [³H]MDL   0.5 nM      5 nM glycine   45 min Scintillation219 insenstive) cortex 105, 519 (1 mM)  0° C. counting (antagonistradioligand) PCP (antagonist rat cerebral [³H]TCP    10 nM     13 nM MK801  120 min Scintillation 257 radioligand) cortex (10 μM) 37° C.counting 5-HT₃ (h) (antagonist human [³H]BRL 43694   0.5 nM   1.15 nMMDL 72222  120 min Scintillation 109 radioligand) recombinant (10 μM) RTcounting (CHO cells) Ca²⁺ channel (L, rat cerebral [³H]initrendipine  0.1 nM   0.18 nM nitrendipine   90 min Scintillation 996dihydropyridine site) cortex (1 μM) RT counting (antagonist radioligand)Ca²⁺ channel (L, rat cerebral [³H]diltiazem    15 nM     52 nM diltiazem 120 min Scintillation 212 diltiazem site) cortex (10 μM) RT counting(benzothiazepines) (antagonist radioligand) Ca²⁺ channel (L, ratcerebral [³H]D888     3 nM      3 nM D 600  120 min Scintillation 194verapamil site) cortex (10 μM) RT counting (phenylalkylamine)(antagonist radioligand) Ca²⁺ channel (N) rat cerebral [¹²⁵I]ω- 0.001 nM0.0007 nM w-conotoxin   30 min Scintillation 259 (antagonist cortexconotoxin GVIA RT counting radioligand) GVIA (10 μM) Potassium Channelhuman [³H]Dofetilide     3 nM    6.6 nM Terfenadine   60 minScintillation 1398 hERG (human)-[3H] recombinant (25 μM) RT countingDofetilide (HEK-293 cells) SK_(Ca) channel rat cerebral [¹²⁵I]apamin0.007 nM  0.007 nM apamin   60 min Scintillation 112 (antagonist cortex(100 μM)  4° C. counting radioligand) Na⁺ channel (site 2) rat cerebral[³H]batrachotoxinin    10 nM     91 nM veratridine   60 minScintillation 28 (antagonist cortex (300 μM) 37° C. countingradioligand) Cl⁻ channel (GABA- rat cerebral [³⁵S]TBPS     3 nM   14.6nM picrotoxinin  120 min Scintillation 136 gated) (antagonist cortex (20μM) RT counting radioligand) Transporters norepinephrine human[³H]nisoxetine     1 nM    2.9 nM desipramine 120 min Scintillation 190transporter (h) recombinant (1 μM)  4° C. counting (antagonist (CHOcells) radioligand) dopamine human [³H]BTCP     4 nM    4.5 nM BTCP  120min Scintillation 190 transporter (h) recombinant (10 μM)  4° C.counting (antagonist (CHO cells) radioligand) GABA transporter ratcerebral [³H]GABA    10 nM   4600 nM GABA   30 min Scintillation 214(antagonist cortex (+10 μM (1 mM) RT counting radioligand) isoguvacine)(+10 μM baclofen) choline transporter human [³H)hemicholinium-3     3 nM   3.9 nM hemicholinium-   60 min Scintillation 648 (CHT1) (h)recombinant 3 RT counting (antagonist (CHO cells) (10 μM) radioligand)5-HT transporter (h) human [³H]imipramine     2 nM    1.7 nM imipramine  60 min Scintillation 566 (antagonist recombinant (10 μM) RT countingradioligand) (CHO cells) Other enzymes MAO-A (antagonist rat cerebral[³H]Ro    10 nM     14 nM clorgyline   60 min Scintillation 36radioligand) cortex 41-1049 (1 μM) 37° C. counting

Table 36 shows enzyme and uptake assay conditions.

TABLE 36 Enzyme and Uptake Assay Conditions Substrate/ MeasuredDetection Assay Source Stimulus/Tracer Incubation Component Method Bibl.Kinases Abl kinase (h) human recombinant ATP + Ulight-TK  60 minphospho-Ulight-TK peptide LANCE 556 (insect cells) peptide RT (100 nM)CaMK2α (h) human recombinant ATP + Ulight-  30 min phospho-Ulight- LANCE647 CGSGSGRPRTSSF AEG RT CGSGSGRPRTSSF AEG (50 nM) CDK2 (h) humanrecombinant ATP + Ulight-  30 min phospho-Ulight- LANCE 469 (cycA)CFFKNIVTPRTPPP RT CFFKNIVTPRTPPP SQGK-amide (50 nM) SQGK-amide ERK2 (h)human recombinant ATP + Ulight-  15 min phospho-Ulight- LANCE 671(P42^(mapk)) (E. coli) CFFKNIVTPRTPPP RT CFFKNIVTPRTPPP SQGK-amide (100nM) SQGK-amide FLT-1 kinase (h) human recombinant ATP + Ulight-TK  15min phospho-Ulight-TK peptide LANCE 650 (VEGFRI) (Sf9 cells) peptide RT(100 nM) Fyn kinase (h) human recombinant ATP + biotinyl-βAβAβ  60 minphospho-biotinyl-βAβAβ HTRF 626 (insect cells) AYQAEENTYDEYEN RTAYQAEENTYDEYEN (2 μM) IRK (h) human recombinant ATP + Ulight-Poly  10min phospho-Ulight-Poly LANCE 467 (InsR) GAT[EAY(1:1:1)]n RTGAT[EAY(1:1:1)]n (50 nM) Lyn A kinase (h) human recombinant ATP +biotinyl-βAβAβ 120 min phospho-biotinyl-βAβAβ HTRF 41 (insect cells)AKVEKIGEGTYGVVYK RT AKVEKIGEGTYGVV (400 nM) YK p38a kinase (h) humanrecombinant ATP + Ulight-  60 min phospho-Ulight- LANCE 620 (E. coli)CFFKNIVTPRTPPP RT CFFKNIVTPRTPPP SQGK-amide SQGK-amide (100 nM) ZAP70kinase (h) human recombinant ATP + biotinyl-  15 minphospho-biotinyl-βAβAβ HTRF 556 (insect cells) βAβAβ RT ADEEEYFIPPADEEEYFIPP (2 μM) Other enzymes COX1(h) human recombinant Arachidonicacid (3 μM) +   3 min Resorufin (oxydized Fluorimetry 1480 ADHP (25 μM)RT ADHP) COX2(h) human recombinant Arachidonic acid (2 μM) +   5 minResorufin (oxydized Fluorimetry 1480 (Sf9 cells) ADHP (25 μM) RT ADHP)5-lipoxygenase (h) human recombinant arachidonic acid  20 min rhodamine123 Fluorimetry 1068 (Sf9 cells) (cytosol) (25 μM) RT 12-lipoxygenase(h) human platelets arachidonic acid   5 min ferric oxidation Photometry472 (4 μM) RT of xylenol orange inducible NOS mouse recombinantL-arginine 120 min NO₂ Photometry 236 (E. coli) (100 μM) 37″C PDE2A1 (h)human recombinant [3H]cAMP + cAMP (2 μM)  20 min [3H]5′AMP Scintillation1399 (Sf9 cells) RT counting PDE3B (h) human recombinant [3H]cAMP + cAMP 20 min [3H]5′AMP Scintillation 1399 (Sf9 cells) (0.5 μM) RT countingPDE4D2 (h) human recombinant [3H]cAMP + cAMP  20 min [³H]5′AMPScintillation 1399 (Sf9 cells) (0.5 μM) RT counting PDE5 (h) humanplatelets [³H]cGMP +  60 min [3H]5′GMP Scintillation 263 (non-selective)cGMP (1 μM) RT counting PDE6 bovine retina [³H]cGMP +  60 min [3H]5'GMPScintillation 306 (non-selective) cGMP (2 μM) RT counting ACE (h) humanrecombinant Abz-FRK(Dnp)-P-OH  30 min Abz-Phe-Arg Fluorimetry 1128 (15μM) 37° C. ACE-2 (h) human recombinant Mca-Tyr-Val-Ala-Asp-  20 min Mcapeptides Fluorimetry 802 (murine cells) Pro-Ala-Lys-(DNP)-OH RT (10 μM)BACE-1 (h) human recombinant Mca-S-E-V-N-L-D-A-E-F  60 minMca-S-E-V-N-L—NH₂ Fluorimetry 462 (β-secretase) (murine cells)R-K(Dnp)-R-R—NH₂ RT (6 μM) caspase-3 (h) human recombinantbenzyloxycarbonyl-Asp-  60 min AFC Fluorimetry 476 (E. coli)Glu-Val-Asp-AFC RT (3.6 μM) caspase-8 (h) human recombinantbenzyloxycarbonyl-Ile-  45 min AFC Fluorimetry 408 (E. coli)Glu-Thr-Asp-AFC (10 μM) 37° C. HIV-1 protease protein viralantranilyl-HIV (75 μM)  40 min N-terminal tripeptide Fluorimetry 244recombinant (E. coli) 37° C. MMP-1 (h) human recombinantDNP-Pro-Cha-Gly-  40 min Cys(Me)-His-Ala- Fluorimetry 342 (E. coli)Cys(Me)-His-Ala-Lys(n- 37° C. Lys(n-Me-Abz)-NH₂ Me-Abz)-NH₂ (10 μM)MMP-2 (h) human recombinant NFF-2  90 min Mca-Arg-Pro-Lys- Fluorimetry297 (10 μM) 37° C. Pro-Tyr-Ala MMP-9 (h) human recombinant NFF-2  90 minMca-Arg-Pro-Lys- Fluorimetry 297 (10 μM) 37° C. Pro-Tyr-Ala guanylylcyclase (h) human recombinant GTP 10 min cGMP HTRF 1076 (activatoreffect) (10 μM) RT (100 μM SNP for control) acetylcholinesterase humanrecombinant Acetylthiocholine (400 μM)  30 min 5 thio 2 nitrobenzoicacid Photometry 63 (h) (HEK-293 cells) RT COMT (catechol-0- porcineliver esculetin  30 min scopoletin Fluorimetry 519 methyl transferase)(1 μM) 37° C. MAO-B (h) human recombinant D-Luciferin  60 min methylester luciferin Luminescence 1134 recombinant derivative 37° C. enzyme(4 μM) xanthine oxidase/ purified xanthine hypoxanthine (10 μM)  10 minO₂ ⁻+ uric acid Photometry 153 superoxide 02- oxidase RT scavenging frombovine milk ATPase(Na⁺/K⁺) porcine cerebral ATP  60 min Pi Photometry 71cortex (2 mM) 37° C. Peptidase, Human Raji cells Glutaryl-Ala-Ala-Phe-4- 30 min Glutaryl-Ala-Ala- Photometry 1352, Metalloproteinase,methoxy-2-naphthylamide 37° C. Phe-4-Methoxy-2- 1353 NeutralEndopeptidase naphthylamide -->4- Methoxy-2- naphthylamine

Analysis and Expression of Results

In Vitro Pharmacology: Binding Assays

The results are expressed as a percent of control specific binding:(Measured specific binding/Control specific binding)×100; and as apercent inhibition of control specific binding: 100−((Measured specificbinding/Control specific binding)×100) obtained in the presence ofNS2-D6.

The IC₅₀ values (concentration causing a half-maximal inhibition ofcontrol specific binding) and Hill coefficients (nH) were determined bynon-linear regression analysis of the competition curves generated withmean replicate values using Hill equation curve fitting:

$Y = {D + \left\lbrack \frac{A\text{∼}D}{1 + \left( {C/C_{50}} \right)^{nH}} \right\rbrack}$

where Y=specific binding, A=left asymptote of the curve, D=rightasymptote of the curve, C=compound concentration, C₅₀=IC₅₀, and nH=slopefactor. This analysis was performed using software developed at Cerep(Hill software) and validated by comparison with data generated by thecommercial software SigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.).The inhibition constants (K_(i)) were calculated using the Cheng Prusoffequation:

$K_{i} = \frac{{IC}_{50}}{\left( {1 + {L/K_{D}}} \right)}$

where L=concentration of radioligand in the assay, and KD=affinity ofthe radioligand for the receptor. A scatchard plot is used to determinethe KD.

In Vitro Pharmacology: Enzyme and Uptake Assays

The results are expressed as a percent of control specific activity:(Measured specific activity/Control specific activity)×100; and as apercent inhibition of control specific activity: 100−((Measured specificactivity/Control specific activity)×100) obtained in the presence ofNS2-D6.

The IC₅₀ values (concentration causing a half-maximal inhibition ofcontrol specific activity), EC₅₀ values (concentration producing ahalf-maximal increase in control basal activity), and Hill coefficients(nH) were determined by non-linear regression analysis of theinhibition/concentration-response curves generated with mean replicatevalues using Hill equation curve fitting:

$Y = {D + \left\lbrack \frac{A\text{∼}D}{1 + \left( {C/C_{50}} \right)^{nH}} \right\rbrack}$

where Y=specific activity, A=left asymptote of the curve, D=rightasymptote of the curve, C=compound concentration, C₅₀=IC₅₀ or EC₅₀, andnH=slope factor. This analysis was performed using software developed atCerep (Hill software) and validated by comparison with data generated bythe commercial software SigmaPlot® 4.0 for Windows® (© 1997 by SPSSInc.).

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We claim: 1-45. (canceled)
 46. A compound of formula VIII-B:

or a pharmaceutically acceptable salt thereof, wherein: each A isindependently hydrogen or deuterium; R¹ is selected from —NH₂, —NHD, or—ND₂; R² is selected from hydrogen or deuterium; R³ and R⁴ areindependently selected from —CH₃, —CH₂D, —CHD₂, or —CD₃; and R⁵ and R⁸are each independently selected from hydrogen or deuterium; providedthat at least one of A, R¹, R², R³, R⁴, R⁵, or R⁸ is or containsdeuterium.
 47. The compound of claim 46, wherein R¹ is —NH₂.
 48. Thecompound of claim 46, at least one instance of A is deuterium.
 49. Thecompound of claim 46, wherein R¹ is —NH₂ and at least one instance of Ais deuterium.
 50. The compound of claim 46, wherein at least two of A,R¹, R², R³, R⁴, R⁵, or R⁸ is or contains deuterium.
 51. The compound ofclaim 46, wherein at least three of A, R¹, R², R³, R⁴, R⁵, or R⁸ is orcontains deuterium.
 52. The compound of claim 46, wherein R² is H. 53.The compound of claim 46, wherein each of R³ and R⁴ is as defined in anentry set forth in the table below: Entry R³ R⁴ i —CH₃ —CH₃ ii —CH₃—CH₂D iii —CH₃ —CHD₂ iv —CH₃ —CD₃ v —CH₂D —CH₃ vi —CH₂D —CH₂D vii —CH₂D—CHD₂ viii —CH₂D —CD₃ ix —CHD₂ —CH₃ x —CHD₂ —CH₂D xi —CHD₂ —CHD₂ xii—CHD₂ —CD₃ xiii —CD₃ —CH₃ xiv —CD₃ —CH₂D xv —CD₃ —CHD₂ xvi —CD₃ —CD₃


54. The compound of claim 53, wherein R³ is —CD₃ and R⁴ is —CD₃.
 55. Thecompound of claim 53, wherein R³ is —CH₃ and R⁴ is —CH₃.
 56. Thecompound of claim 46, wherein R⁵ is D.
 57. The compound of claim 46,wherein R⁵ is H.
 58. The compound of claim 46, wherein R⁸ is D.
 59. Thecompound of claim 46, wherein R⁸ is H.
 60. A pharmaceutical compositioncomprising the compound of claim 46, or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable adjuvant, carrier, orvehicle.
 61. A composition comprising a compound of claim 46, whereineach position of deuterium enrichment in the compound comprisesdeuterium in an amount of about 50% or greater.
 62. The composition ofclaim 46, wherein each position of deuterium enrichment in the compoundcomprises deuterium in an amount of about 80% or greater.
 63. Thecomposition of claim 53, wherein each position of deuterium enrichmentin the compound comprises deuterium in an amount of 90% or greater. 64.The composition of claim 54, wherein each position of deuteriumenrichment in the compound comprises deuterium in an amount of 90% orgreater.
 65. The composition of claim 54, wherein each position ofdeuterium enrichment in the compound comprises deuterium in an amount of95% or greater.